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1
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Koike A, Brindley PJ. CRISPR/Cas genome editing, functional genomics, and diagnostics for parasitic helminths. Int J Parasitol 2025:S0020-7519(25)00092-X. [PMID: 40348052 DOI: 10.1016/j.ijpara.2025.05.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2025] [Revised: 04/30/2025] [Accepted: 05/04/2025] [Indexed: 05/14/2025]
Abstract
Functional genomics using CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated endonuclease)-based approaches has revolutionized biomedical sciences. Gene editing is also widespread in parasitology generally and its use is increasing in studies on helminths including flatworm and roundworm parasites. Here, we survey the progress, specifically with experimental CRISPR-facilitated functional genomics to investigate helminth biology and pathogenesis, and also with the burgeoning use of CRISPR-based methods to assist in diagnosis of helminth infections. We also provide an historical timeline of the introduction and uses of CRISPR in helminth species to date.
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Affiliation(s)
- Akito Koike
- Department of Microbiology, Immunology & Tropical Medicine, School of Medicine & Health Sciences, George Washington University, Washington, D.C. 20037, USA
| | - Paul J Brindley
- Department of Microbiology, Immunology & Tropical Medicine, School of Medicine & Health Sciences, George Washington University, Washington, D.C. 20037, USA.
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2
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Li J, Cui J, Li X, Zhu D, Chen Z, Huang X, Wang Y, Wu Q, Tian Y. TMBIM-2 orchestrates systemic mitochondrial stress response via facilitating Ca2+ oscillations. J Cell Biol 2025; 224:e202408050. [PMID: 40100072 PMCID: PMC11917168 DOI: 10.1083/jcb.202408050] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2024] [Revised: 12/11/2024] [Accepted: 02/09/2025] [Indexed: 03/20/2025] Open
Abstract
Neuronal mitochondrial function is critical for orchestrating inter-tissue communication essential for overall fitness. Despite its significance, the molecular mechanism underlying the impact of prolonged mitochondrial stresses on neuronal activity and how they orchestrate metabolism and aging remains elusive. Here, we identified the evolutionarily conserved transmembrane protein XBX-6/TMBIM-2 as a key mediator in the neuronal-to-intestinal mitochondrial unfolded protein response (UPRmt). Our investigations reveal that intrinsic neuronal mitochondrial stress triggers spatiotemporal Ca2+ oscillations in a TMBIM-2-dependent manner through the Ca2+ efflux pump MCA-3. Notably, persistent Ca2+ oscillations at synapses of ADF neurons are critical for facilitating serotonin release and the subsequent activation of the neuronal-to-intestinal UPRmt. TMBIM2 expression diminishes with age; however, its overexpression counteracts the age-related decline in aversive learning behavior and extends the lifespan of Caenorhabditis elegans. These findings underscore the intricate integration of chronic neuronal mitochondrial stress into neurotransmission processes via TMBIM-2-dependent Ca2+ equilibrium, driving metabolic adaptation and behavioral changes for the regulation of aging.
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Affiliation(s)
- Jiasheng Li
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Jimeng Cui
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Xinyu Li
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Di Zhu
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Zhenhua Chen
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Xiahe Huang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
| | - Yingchun Wang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Qingfeng Wu
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Ye Tian
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
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3
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Shanley HT, Wang T, Taki AC, Byrne JJ, Chang BCH, Sleebs BE, Gasser RB. Advances in Anthelmintic Target Identification. Int J Mol Sci 2025; 26:3738. [PMID: 40332360 PMCID: PMC12028019 DOI: 10.3390/ijms26083738] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2025] [Revised: 04/11/2025] [Accepted: 04/13/2025] [Indexed: 05/08/2025] Open
Abstract
Parasitic nematodes pose a significant threat to human and animal health, causing widespread morbidity and substantial socioeconomic losses globally. Despite the utility of anthelmintic drugs in parasite control, the emergence of widespread resistance necessitates the discovery of novel interventions. Advances through the use of whole-organism phenotypic screening have identified some promising nematocidal compounds, including nemacol, tolfenpyrad, UMW-9729, and ABX464. This article summarises efforts in this discovery, with a focus on Haemonchus contortus and Caenorhabditis elegans as model nematodes, and discusses approaches used for drug target deconvolution, including proteomic, chemical and genetic/genomic techniques. Stability-based proteomic assays, such as thermal proteome profiling, have been useful for identifying protein targets for these compounds, shedding light on their mechanisms of action. However, challenges remain in extrapolating findings from C. elegans to parasitic nematodes, emphasising the need for validation studies. Understanding drug-target interactions in nematodes is critical for developing next-generation anthelmintics and for mitigating the growing resistance challenge. This review outlines recent progress in this area and discusses future directions in target validation and anthelmintic development to support parasite control programmes.
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Affiliation(s)
- Harrison T. Shanley
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia; (H.T.S.); (T.W.); (A.C.T.); (J.J.B.); (B.C.H.C.)
| | - Tao Wang
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia; (H.T.S.); (T.W.); (A.C.T.); (J.J.B.); (B.C.H.C.)
| | - Aya C. Taki
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia; (H.T.S.); (T.W.); (A.C.T.); (J.J.B.); (B.C.H.C.)
| | - Joseph J. Byrne
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia; (H.T.S.); (T.W.); (A.C.T.); (J.J.B.); (B.C.H.C.)
| | - Bill C. H. Chang
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia; (H.T.S.); (T.W.); (A.C.T.); (J.J.B.); (B.C.H.C.)
| | - Brad E. Sleebs
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia; (H.T.S.); (T.W.); (A.C.T.); (J.J.B.); (B.C.H.C.)
- Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia
| | - Robin B. Gasser
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia; (H.T.S.); (T.W.); (A.C.T.); (J.J.B.); (B.C.H.C.)
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4
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Nievergelt AP. Genome editing in the green alga Chlamydomonas: past, present practice and future prospects. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2025; 122:e70140. [PMID: 40186543 PMCID: PMC11971955 DOI: 10.1111/tpj.70140] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/30/2024] [Revised: 03/20/2025] [Accepted: 03/26/2025] [Indexed: 04/07/2025]
Abstract
The green alga Chlamydomonas is an important and versatile model organism for research topics ranging from photosynthesis and metabolism, cilia, and basal bodies to cellular communication and the cellular cycle and is of significant interest for green bioengineering processes. The genome in this unicellular green alga is contained in 17 haploid chromosomes and codes for 16 883 protein coding genes. Functional genomics, as well as biotechnological applications, rely on the ability to remove, add, and change these genes in a controlled and efficient manner. In this review, the history of gene editing in Chlamydomonas is put in the context of the wider developments in genetics to demonstrate how many of the key developments to engineer these algae follow the global trends and the availability of technology. Building on this background, an overview of the state of the art in Chlamydomonas engineering is given, focusing primarily on the practical aspects while giving examples of recent applications. Commonly encountered Chlamydomonas-specific challenges, recent developments, and community resources are presented, and finally, a comprehensive discussion on the emergence and evolution of CRISPR/Cas-based precision gene editing is given. An outline of possible future paths for gene editing based on current global trends in genetic engineering and tools for gene editing is presented.
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Affiliation(s)
- Adrian P. Nievergelt
- Max Planck Institute of Molecular Cell Biology and GeneticsPfotenhauerstraße 108Dresden01307Germany
- Max Planck Institute of Molecular Plant PhysiologyAm Mühlenberg 1Potsdam14476Germany
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5
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Verschuren J, van Schendel R, van Bostelen I, Verkennis AEE, Knipscheer P, Tijsterman M. FAN1-mediated translesion synthesis and POLQ/HELQ-mediated end joining generate interstrand crosslink-induced mutations. Nat Commun 2025; 16:2495. [PMID: 40082407 PMCID: PMC11906846 DOI: 10.1038/s41467-025-57764-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2024] [Accepted: 03/04/2025] [Indexed: 03/16/2025] Open
Abstract
To counteract the damaging effects of DNA interstrand crosslinks (ICLs), cells have evolved various specialized ICL repair pathways. However, how ICL repair impacts genetic integrity remains incompletely understood. Here, we determined the mutagenic consequences of psoralen ICL repair in the animal model C. elegans and identify two mutagenic repair mechanisms: (i) translesion synthesis through POLH and REV1/3-mediated bypass, leading to single nucleotide polymorphisms (SNVs), and (ii) end joining via POLQ or HELQ action resulting in deletions. While we found no role for the Fanconi anemia genes FANCD2 and FANCI, disruption of TRAIP, which triggers unloading of the CMG helicase at sites of blocked replication, led to a strikingly altered repair profile, suggesting a role for DNA replication in the etiology of ICL-induced deletions. TRAIP deficiency did not affect SNV formation; instead, we found these SNVs to depend on the functionality of the Fanconi anemia-associated nuclease FAN1.
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Affiliation(s)
- Jip Verschuren
- Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
| | - Robin van Schendel
- Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
| | - Ivo van Bostelen
- Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
| | - Alex E E Verkennis
- Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands
| | - Puck Knipscheer
- Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
- Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands
| | - Marcel Tijsterman
- Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
- Institute of Biology Leiden, Leiden University, Leiden, The Netherlands.
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6
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Prevo B, Cheerambathur DK, Earnshaw WC, Desai A. Kinetochore dynein is sufficient to biorient chromosomes and remodel the outer kinetochore. Nat Commun 2024; 15:9085. [PMID: 39433738 PMCID: PMC11494143 DOI: 10.1038/s41467-024-52964-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2024] [Accepted: 09/25/2024] [Indexed: 10/23/2024] Open
Abstract
Multiple microtubule-directed activities concentrate on mitotic chromosomes to ensure their faithful segregation. These include couplers and dynamics regulators localized at the kinetochore, the microtubule interface built on centromeric chromatin, as well as motor proteins recruited to kinetochores and chromatin. Here, we describe an in vivo approach in the C. elegans one-cell embryo in which removal of the major microtubule-directed activities on mitotic chromosomes is compared to the selective presence of individual activities. Our approach reveals that the kinetochore dynein module, comprised of cytoplasmic dynein and its kinetochore-specific adapters, is sufficient to biorient chromosomes; by contrast, this module is unable to support congression. In coordination with orientation, the dynein module directs removal of outermost kinetochore components, including dynein itself, independently of the other microtubule-directed activities and kinetochore-localized protein phosphatase 1. These observations indicate that the kinetochore dynein module is sufficient to biorient chromosomes and to direct remodeling of the outer kinetochore in a microtubule attachment state-sensitive manner.
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Affiliation(s)
- Bram Prevo
- Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.
- Ludwig Institute for Cancer Research, La Jolla, CA, USA.
| | | | - William C Earnshaw
- Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK
| | - Arshad Desai
- Ludwig Institute for Cancer Research, La Jolla, CA, USA.
- Department of Cell and Developmental Biology, School of Biological Sciences, University of California San Diego, La Jolla, CA, USA.
- Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.
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7
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Wang Z, Zhang Q, Jiang Y, Zhou J, Tian Y. ASI-RIM neuronal axis regulates systemic mitochondrial stress response via TGF-β signaling cascade. Nat Commun 2024; 15:8997. [PMID: 39426950 PMCID: PMC11490647 DOI: 10.1038/s41467-024-53093-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2024] [Accepted: 09/30/2024] [Indexed: 10/21/2024] Open
Abstract
Morphogens play a critical role in coordinating stress adaptation and aging across tissues, yet their involvement in neuronal mitochondrial stress responses and systemic effects remains unclear. In this study, we reveal that the transforming growth factor beta (TGF-β) DAF-7 is pivotal in mediating the intestinal mitochondrial unfolded protein response (UPRmt) in Caenorhabditis elegans under neuronal mitochondrial stress. Two ASI sensory neurons produce DAF-7, which targets DAF-1/TGF-β receptors on RIM interneurons to orchestrate a systemic UPRmt response. Remarkably, inducing mitochondrial stress specifically in ASI neurons activates intestinal UPRmt, extends lifespan, enhances pathogen resistance, and reduces both brood size and body fat levels. Furthermore, dopamine positively regulates this UPRmt activation, while GABA acts as a systemic suppressor. This study uncovers the intricate mechanisms of systemic mitochondrial stress regulation, emphasizing the vital role of TGF-β in metabolic adaptations that are crucial for organismal fitness and aging during neuronal mitochondrial stress.
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Affiliation(s)
- Zihao Wang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 100101, Beijing, China
- University of Chinese Academy of Sciences, 100093, Beijing, China
| | - Qian Zhang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 100101, Beijing, China
| | - Yayun Jiang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 100101, Beijing, China
- University of Chinese Academy of Sciences, 100093, Beijing, China
| | - Jun Zhou
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 100101, Beijing, China
- University of Chinese Academy of Sciences, 100093, Beijing, China
| | - Ye Tian
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 100101, Beijing, China.
- University of Chinese Academy of Sciences, 100093, Beijing, China.
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8
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Shanley HT, Taki AC, Nguyen N, Wang T, Byrne JJ, Ang CS, Leeming MG, Williamson N, Chang BCH, Jabbar A, Sleebs BE, Gasser RB. Comparative structure activity and target exploration of 1,2-diphenylethynes in Haemonchus contortus and Caenorhabditis elegans. Int J Parasitol Drugs Drug Resist 2024; 25:100534. [PMID: 38554597 PMCID: PMC10992699 DOI: 10.1016/j.ijpddr.2024.100534] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2024] [Revised: 03/14/2024] [Accepted: 03/17/2024] [Indexed: 04/01/2024]
Abstract
Infections and diseases caused by parasitic nematodes have a major adverse impact on the health and productivity of animals and humans worldwide. The control of these parasites often relies heavily on the treatment with commercially available chemical compounds (anthelmintics). However, the excessive or uncontrolled use of these compounds in livestock animals has led to major challenges linked to drug resistance in nematodes. Therefore, there is a need to develop new anthelmintics with novel mechanism(s) of action. Recently, we identified a small molecule, designated UMW-9729, with nematocidal activity against the free-living model organism Caenorhabditis elegans. Here, we evaluated UMW-9729's potential as an anthelmintic in a structure-activity relationship (SAR) study in C. elegans and the highly pathogenic, blood-feeding Haemonchus contortus (barber's pole worm), and explored the compound-target relationship using thermal proteome profiling (TPP). First, we synthesised and tested 25 analogues of UMW-9729 for their nematocidal activity in both H. contortus (larvae and adults) and C. elegans (young adults), establishing a preliminary nematocidal pharmacophore for both species. We identified several compounds with marked activity against either H. contortus or C. elegans which had greater efficacy than UMW-9729, and found a significant divergence in compound bioactivity between these two nematode species. We also identified a UMW-9729 analogue, designated 25, that moderately inhibited the motility of adult female H. contortus in vitro. Subsequently, we inferred three H. contortus proteins (HCON_00134350, HCON_00021470 and HCON_00099760) and five C. elegans proteins (F30A10.9, F15B9.8, B0361.6, DNC-4 and UNC-11) that interacted directly with UMW-9729; however, no conserved protein target was shared between the two nematode species. Future work aims to extend the SAR investigation in these and other parasitic nematode species, and validate individual proteins identified here as possible targets of UMW-9729. Overall, the present study evaluates this anthelmintic candidate and highlights some challenges associated with early anthelmintic investigation.
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Affiliation(s)
- Harrison T Shanley
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, Victoria, 3010, Australia; Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3052, Australia
| | - Aya C Taki
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, Victoria, 3010, Australia
| | - Nghi Nguyen
- Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3052, Australia
| | - Tao Wang
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, Victoria, 3010, Australia
| | - Joseph J Byrne
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, Victoria, 3010, Australia
| | - Ching-Seng Ang
- Melbourne Mass Spectrometry and Proteomics Facility, The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, 3010, Australia
| | - Michael G Leeming
- Melbourne Mass Spectrometry and Proteomics Facility, The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, 3010, Australia
| | - Nicholas Williamson
- Melbourne Mass Spectrometry and Proteomics Facility, The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, 3010, Australia
| | - Bill C H Chang
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, Victoria, 3010, Australia
| | - Abdul Jabbar
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, Victoria, 3010, Australia
| | - Brad E Sleebs
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, Victoria, 3010, Australia; Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3052, Australia.
| | - Robin B Gasser
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Science, The University of Melbourne, Parkville, Victoria, 3010, Australia.
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9
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Song B, Bae S. Genome editing using CRISPR, CAST, and Fanzor systems. Mol Cells 2024; 47:100086. [PMID: 38909984 PMCID: PMC11278801 DOI: 10.1016/j.mocell.2024.100086] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2024] [Revised: 06/14/2024] [Accepted: 06/18/2024] [Indexed: 06/25/2024] Open
Abstract
Genetic engineering technologies are essential not only for basic science but also for generating animal models for therapeutic applications. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system, derived from adapted prokaryotic immune responses, has led to unprecedented advancements in the field of genome editing because of its ability to precisely target and edit genes in a guide RNA-dependent manner. The discovery of various types of CRISPR-Cas systems, such as CRISPR-associated transposons (CASTs), has resulted in the development of novel genome editing tools. Recently, research has expanded to systems associated with obligate mobile element guided activity (OMEGA) RNAs, including ancestral CRISPR-Cas and eukaryotic Fanzor systems, which are expected to complement the conventional CRISPR-Cas systems. In this review, we briefly introduce the features of various CRISPR-Cas systems and their application in diverse animal models.
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Affiliation(s)
- Beomjong Song
- Department of Anatomy, College of Medicine, Soonchunhyang University, Cheonan 33151, Republic of Korea.
| | - Sangsu Bae
- Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, Republic of Korea; Medical Research Center of Genomic Medicine Institute, Seoul National University College of Medicine, Seoul 03080, Republic of Korea; Cancer Research Institute, Seoul National University College of Medicine, Seoul 03080, Republic of Korea; Institute of Molecular Biology and Genetics, Seoul National University, Seoul 08826, Republic of Korea.
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10
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Houston J, Vissotsky C, Deep A, Hakozaki H, Crews E, Oegema K, Corbett KD, Lara-Gonzalez P, Kim T, Desai A. Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to C. elegans kinetochores. J Cell Biol 2024; 223:e202402036. [PMID: 38578284 PMCID: PMC10996584 DOI: 10.1083/jcb.202402036] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2024] [Revised: 03/21/2024] [Accepted: 03/25/2024] [Indexed: 04/06/2024] Open
Abstract
During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide repeat (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1's TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.
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Affiliation(s)
- Jack Houston
- Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA, USA
- Department of Cell and Developmental Biology, School of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
- Ludwig Institute for Cancer Research, La Jolla, CA, USA
| | | | - Amar Deep
- Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA
| | - Hiroyuki Hakozaki
- Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA
| | - Enice Crews
- Department of Cell and Developmental Biology, School of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
| | - Karen Oegema
- Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA, USA
- Department of Cell and Developmental Biology, School of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
- Ludwig Institute for Cancer Research, La Jolla, CA, USA
- Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA
| | - Kevin D. Corbett
- Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA, USA
- Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA
- Department of Molecular Biology, School of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
| | - Pablo Lara-Gonzalez
- Ludwig Institute for Cancer Research, La Jolla, CA, USA
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA
| | - Taekyung Kim
- Ludwig Institute for Cancer Research, La Jolla, CA, USA
- Department of Biology Education, Pusan National University, Busan, Republic of Korea
| | - Arshad Desai
- Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA, USA
- Department of Cell and Developmental Biology, School of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
- Ludwig Institute for Cancer Research, La Jolla, CA, USA
- Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA
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11
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Houston J, Vissotsky C, Deep A, Hakozaki H, Crews E, Oegema K, Corbett KD, Lara-Gonzalez P, Kim T, Desai A. Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to C. elegans kinetochores. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.02.09.579536. [PMID: 38370671 PMCID: PMC10871365 DOI: 10.1101/2024.02.09.579536] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/20/2024]
Abstract
During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1's TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.
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Affiliation(s)
- Jack Houston
- Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, California 92093, USA
- Department of Cell & Developmental Biology, School of Biological Sciences, University of California San Diego, La Jolla, California 92093, USA
- Ludwig Institute for Cancer Research, La Jolla, California 92093, USA
| | | | - Amar Deep
- Department of Cellular & Molecular Medicine, University of California San Diego, La Jolla, California 92093, USA
| | - Hiro Hakozaki
- Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA
| | - Enice Crews
- Department of Cell & Developmental Biology, School of Biological Sciences, University of California San Diego, La Jolla, California 92093, USA
| | - Karen Oegema
- Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, California 92093, USA
- Department of Cell & Developmental Biology, School of Biological Sciences, University of California San Diego, La Jolla, California 92093, USA
- Ludwig Institute for Cancer Research, La Jolla, California 92093, USA
- Department of Cellular & Molecular Medicine, University of California San Diego, La Jolla, California 92093, USA
| | - Kevin D. Corbett
- Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, California 92093, USA
- Department of Cellular & Molecular Medicine, University of California San Diego, La Jolla, California 92093, USA
- Department of Molecular Biology, School of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA
| | - Pablo Lara-Gonzalez
- Ludwig Institute for Cancer Research, La Jolla, California 92093, USA
- Department of Developmental & Cell Biology, University of California Irvine, Irvine, CA 92697, USA
| | - Taekyung Kim
- Ludwig Institute for Cancer Research, La Jolla, California 92093, USA
- Department of Biology Education, Pusan National University, Busan 46241, Republic of Korea
| | - Arshad Desai
- Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, California 92093, USA
- Department of Cell & Developmental Biology, School of Biological Sciences, University of California San Diego, La Jolla, California 92093, USA
- Ludwig Institute for Cancer Research, La Jolla, California 92093, USA
- Department of Cellular & Molecular Medicine, University of California San Diego, La Jolla, California 92093, USA
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12
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Yamamoto S, Kanca O, Wangler MF, Bellen HJ. Integrating non-mammalian model organisms in the diagnosis of rare genetic diseases in humans. Nat Rev Genet 2024; 25:46-60. [PMID: 37491400 DOI: 10.1038/s41576-023-00633-6] [Citation(s) in RCA: 31] [Impact Index Per Article: 31.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/26/2023] [Indexed: 07/27/2023]
Abstract
Next-generation sequencing technology has rapidly accelerated the discovery of genetic variants of interest in individuals with rare diseases. However, showing that these variants are causative of the disease in question is complex and may require functional studies. Use of non-mammalian model organisms - mainly fruitflies (Drosophila melanogaster), nematode worms (Caenorhabditis elegans) and zebrafish (Danio rerio) - enables the rapid and cost-effective assessment of the effects of gene variants, which can then be validated in mammalian model organisms such as mice and in human cells. By probing mechanisms of gene action and identifying interacting genes and proteins in vivo, recent studies in these non-mammalian model organisms have facilitated the diagnosis of numerous genetic diseases and have enabled the screening and identification of therapeutic options for patients. Studies in non-mammalian model organisms have also shown that the biological processes underlying rare diseases can provide insight into more common mechanisms of disease and the biological functions of genes. Here, we discuss the opportunities afforded by non-mammalian model organisms, focusing on flies, worms and fish, and provide examples of their use in the diagnosis of rare genetic diseases.
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Affiliation(s)
- Shinya Yamamoto
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
- Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA
- Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA
| | - Oguz Kanca
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
- Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA
| | - Michael F Wangler
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
- Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA.
| | - Hugo J Bellen
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
- Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, USA.
- Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA.
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13
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Chen Y, Arlt VM, Stürzenbaum SR. MosSCI-mediated exogenous gene expression is modulated by genomic positioning. Biotechnol J 2023; 18:e2300062. [PMID: 37177911 DOI: 10.1002/biot.202300062] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2023] [Revised: 05/09/2023] [Accepted: 05/11/2023] [Indexed: 05/15/2023]
Abstract
Although the Mos1-mediated single-copy insertion (MosSCI) technique has been widely used to generate stable transgenic Caenorhabditis elegans strains, the link between stability of expression and integration site still needs to be explored. Here, experimental evidence is provided that transgenes are not able to match the level of transcription of their native counterpart, and that insertions at certain locations can result in an external stress-mediated increase in expression. Insertion site ttTi5605 on chromosome II was shown to be a superior location, at least when introducing reproduction related genes. Thus, this study provides a reference for the selection of an optimal site for MosSCI which provides acceptable expression performance whilst minimizing undesirable secondary effects.
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Affiliation(s)
- Yuzhi Chen
- Department of Analytical, Environmental and Forensic Sciences, School of Cancer & Pharmaceutical Sciences, Faculty of Life Sciences and Medicine, King's College London, London, UK
| | - Volker M Arlt
- Toxicology Department, GAB Consulting GmbH, Heidelberg, Germany
| | - Stephen R Stürzenbaum
- Department of Analytical, Environmental and Forensic Sciences, School of Cancer & Pharmaceutical Sciences, Faculty of Life Sciences and Medicine, King's College London, London, UK
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14
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Villao L, Chávez T, Pacheco R, Sánchez E, Bonilla J, Santos E. Genetic improvement in Musa through modern biotechnological methods. BIONATURA 2023. [DOI: 10.21931/rb/2023.08.01.20] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/12/2023] Open
Abstract
Bananas, one of the most valued fruits worldwide, are produced in more than 135 countries in the tropics and subtropics for local consumption and export due to their tremendous nutritional value and ease of access.
The genetic improvement of commercial crops is a crucial strategy for managing pests or other diseases and abiotic stress factors. Although conventional breeding has developed new hybrids with highly productive or agronomic performance characteristics, in some banana cultivars, due to the high level of sterility, the traditional breeding strategy is hampered. Therefore, modern biotechniques have been developed in a banana for genetic improvement. In vitro, culture techniques have been a basis for crop micropropagation for elite banana varieties and the generation of methods for genetic modification. This review includes topics of great interest for improving bananas and their products worldwide, from their origins to the different improvement alternatives.
Keywords. Banana, genetic improvement, pest management, diseases, abiotic stress factors.
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Affiliation(s)
- L, Villao
- Escuela Superior Politécnica del Litoral, ESPOL, Biotechnological Research Center of Ecuador, Gustavo Galindo Campus Km. 30.5 Vía Perimetral, PO Box 09-01-5863, Guayaquil, Ecuador
| | - T, Chávez
- Escuela Superior Politécnica del Litoral, ESPOL, Biotechnological Research Center of Ecuador, Gustavo Galindo Campus Km. 30.5 Vía Perimetral, PO Box 09-01-5863, Guayaquil, Ecuador
| | - R, Pacheco
- Escuela Superior Politécnica del Litoral, ESPOL, Biotechnological Research Center of Ecuador, Gustavo Galindo Campus Km. 30.5 Vía Perimetral, PO Box 09-01-5863, Guayaquil, Ecuador
| | - E. Sánchez
- Escuela Superior Politécnica del Litoral, ESPOL, Biotechnological Research Center of Ecuador, Gustavo Galindo Campus Km. 30.5 Vía Perimetral, PO Box 09-01-5863, Guayaquil, Ecuador; Escuela Superior Politécnica del Litoral, ESPOL, Faculty of Life Sciences, Gustavo Galindo Campus Km. 30.5 Vía Perimetral, PO Box 09-01-5863, Guayaquil, Ecuador
| | - J. Bonilla
- Escuela Superior Politécnica del Litoral, ESPOL, Biotechnological Research Center of Ecuador, Gustavo Galindo Campus Km. 30.5 Vía Perimetral, PO Box 09-01-5863, Guayaquil, Ecuador ; Escuela Superior Politécnica del Litoral, ESPOL, Faculty of Life Sciences, Gustavo Galindo Campus Km. 30.5 Vía Perimetral, PO Box 09-01-5863, Guayaquil, Ecuador
| | - E. Santos
- Escuela Superior Politécnica del Litoral, ESPOL, Biotechnological Research Center of Ecuador, Gustavo Galindo Campus Km. 30.5 Vía Perimetral, PO Box 09-01-5863, Guayaquil, Ecuador ; Escuela Superior Politécnica del Litoral, ESPOL, Faculty of Life Sciences, Gustavo Galindo Campus Km. 30.5 Vía Perimetral, PO Box 09-01-5863, Guayaquil, Ecuador
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15
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A comprehensive evaluation of stable expression "hot spot" in the ScltI gene of Chinese hamster ovary cells. Appl Microbiol Biotechnol 2023; 107:1299-1309. [PMID: 36707420 DOI: 10.1007/s00253-023-12383-w] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2022] [Revised: 01/06/2023] [Accepted: 01/09/2023] [Indexed: 01/29/2023]
Abstract
The Chinese hamster ovary (CHO) cell is the most widely used biopharmaceutical expression system, but its long-term expression is unstable. This issue can be effectively addressed by site-specific integration of exogenous genes into the genome. Therefore, exogenous protein sites with stable expression in the CHO cell genome must be identified. CRISPR/Cas9 technology was used in this study to integrate various exogenous genes into the ScltI site as a "hot spot" at the CHO-K1 cell genome NW_003614095.1, and the stability and adaptability of exogenous genes expressed at the site were investigated. Flow cytometry sorting technology was used to obtain positive monoclonal cell lines that expressed either intracellular protein green fluorescent protein (EGFP) or secretory protein human serum albumin (HSA). For 60 passages, the positive monoclonal cell lines' cell growth cycles and exogenous protein expression were both observed. The results demonstrated that integrating the gene encoding exogenous proteins into the ScltI site had no effect on cell growth. The fluorescence intensity of EGFP was similar after 60 passages, and the expression of HSA increased slightly. Additionally, the super-monomeric protein VWF hydrolase (ADAMTS13) (190 kDa), human coagulation factor VII (FVII) (55 kDa), and interferon α2b (12 kDa) were integrated into the ScltI site for expression. In conclusion, the site located in the first exon of the ScltI gene within the CHO-K1 cell genome NW_003614095.1 is an ideal "hot spot" for the stable expression of various exogenous proteins. KEY POINTS: • The site-specific integration strategy of an exogenous gene in CHO cells was established for the ScltI site. • The genes for EGFP and HSA were site-directed integrated and stably expressed at the ScltI site. • The ScltI site fulfills the expression of exogenous proteins of different molecular weight sizes (15-190 kDa).
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16
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Kim HM, Hong Y, Chen J. A Decade of CRISPR-Cas Gnome Editing in C. elegans. Int J Mol Sci 2022; 23:ijms232415863. [PMID: 36555505 PMCID: PMC9781986 DOI: 10.3390/ijms232415863] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2022] [Revised: 12/05/2022] [Accepted: 12/05/2022] [Indexed: 12/23/2022] Open
Abstract
CRISPR-Cas allows us to introduce desired genome editing, including mutations, epitopes, and deletions, with unprecedented efficiency. The development of CRISPR-Cas has progressed to such an extent that it is now applicable in various fields, with the help of model organisms. C. elegans is one of the pioneering animals in which numerous CRISPR-Cas strategies have been rapidly established over the past decade. Ironically, the emergence of numerous methods makes the choice of the correct method difficult. Choosing an appropriate selection or screening approach is the first step in planning a genome modification. This report summarizes the key features and applications of CRISPR-Cas methods using C. elegans, illustrating key strategies. Our overview of significant advances in CRISPR-Cas will help readers understand the current advances in genome editing and navigate various methods of CRISPR-Cas genome editing.
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17
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Ding Q, Ren X, Li R, Chan L, Ho VWS, Bi Y, Xie D, Zhao Z. Highly efficient transgenesis with miniMos in Caenorhabditis briggsae. G3 (BETHESDA, MD.) 2022; 12:jkac254. [PMID: 36171682 PMCID: PMC9713419 DOI: 10.1093/g3journal/jkac254] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 07/12/2022] [Accepted: 08/29/2022] [Indexed: 06/13/2023]
Abstract
Caenorhabditis briggsae as a companion species for Caenorhabditis elegans has played an increasingly important role in study of evolution of development and genome and gene regulation. Aided by the isolation of its sister spices, it has recently been established as a model for speciation study. To take full advantage of the species for comparative study, an effective transgenesis method especially those with single-copy insertion is important for functional comparison. Here, we improved a transposon-based transgenesis methodology that had been originally developed in C. elegans but worked marginally in C. briggsae. By incorporation of a heat shock step, the transgenesis efficiency in C. briggsae with a single-copy insertion is comparable to that in C. elegans. We used the method to generate 54 independent insertions mostly consisting of a mCherry tag over the C. briggsae genome. We demonstrated the use of the tags in identifying interacting loci responsible for hybrid male sterility between C. briggsae and Caenorhabditis nigoni when combined with the GFP tags we generated previously. Finally, we demonstrated that C. briggsae tolerates the C. elegans toxin, PEEL-1, but not SUP-35, making the latter a potential negative selection marker against extrachromosomal array.
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Affiliation(s)
- Qiutao Ding
- Department of Biology, Hong Kong Baptist University, Hong Kong, China
| | - Xiaoliang Ren
- Department of Biology, Hong Kong Baptist University, Hong Kong, China
| | - Runsheng Li
- Department of Biology, Hong Kong Baptist University, Hong Kong, China
- Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Hong Kong, China
| | - Luyan Chan
- Department of Biology, Hong Kong Baptist University, Hong Kong, China
| | - Vincy W S Ho
- Department of Biology, Hong Kong Baptist University, Hong Kong, China
| | - Yu Bi
- Department of Biology, Hong Kong Baptist University, Hong Kong, China
| | - Dongying Xie
- Department of Biology, Hong Kong Baptist University, Hong Kong, China
| | - Zhongying Zhao
- Department of Biology, Hong Kong Baptist University, Hong Kong, China
- State Key Laboratory of Environmental and Biological Analysis, Hong Kong Baptist University, Hong Kong SAR, China
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18
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Kim J, Jimenez DS, Ragipani B, Zhang B, Street LA, Kramer M, Albritton SE, Winterkorn LH, Morao AK, Ercan S. Condensin DC loads and spreads from recruitment sites to create loop-anchored TADs in C. elegans. eLife 2022; 11:e68745. [PMID: 36331876 PMCID: PMC9635877 DOI: 10.7554/elife.68745] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2021] [Accepted: 10/23/2022] [Indexed: 11/06/2022] Open
Abstract
Condensins are molecular motors that compact DNA via linear translocation. In Caenorhabditis elegans, the X-chromosome harbors a specialized condensin that participates in dosage compensation (DC). Condensin DC is recruited to and spreads from a small number of recruitment elements on the X-chromosome (rex) and is required for the formation of topologically associating domains (TADs). We take advantage of autosomes that are largely devoid of condensin DC and TADs to address how rex sites and condensin DC give rise to the formation of TADs. When an autosome and X-chromosome are physically fused, despite the spreading of condensin DC into the autosome, no TAD was created. Insertion of a strong rex on the X-chromosome results in the TAD boundary formation regardless of sequence orientation. When the same rex is inserted on an autosome, despite condensin DC recruitment, there was no spreading or features of a TAD. On the other hand, when a 'super rex' composed of six rex sites or three separate rex sites are inserted on an autosome, recruitment and spreading of condensin DC led to the formation of TADs. Therefore, recruitment to and spreading from rex sites are necessary and sufficient for recapitulating loop-anchored TADs observed on the X-chromosome. Together our data suggest a model in which rex sites are both loading sites and bidirectional barriers for condensin DC, a one-sided loop-extruder with movable inactive anchor.
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Affiliation(s)
- Jun Kim
- Department of Biology and Center for Genomics and Systems Biology, New York UniversityNew YorkUnited States
| | - David S Jimenez
- Department of Biology and Center for Genomics and Systems Biology, New York UniversityNew YorkUnited States
| | - Bhavana Ragipani
- Department of Biology and Center for Genomics and Systems Biology, New York UniversityNew YorkUnited States
| | - Bo Zhang
- UCSF HSWSan FranciscoUnited States
| | - Lena A Street
- Department of Biology and Center for Genomics and Systems Biology, New York UniversityNew YorkUnited States
| | - Maxwell Kramer
- Department of Biology and Center for Genomics and Systems Biology, New York UniversityNew YorkUnited States
| | - Sarah E Albritton
- Department of Biology and Center for Genomics and Systems Biology, New York UniversityNew YorkUnited States
| | - Lara H Winterkorn
- Department of Biology and Center for Genomics and Systems Biology, New York UniversityNew YorkUnited States
| | - Ana K Morao
- Department of Biology and Center for Genomics and Systems Biology, New York UniversityNew YorkUnited States
| | - Sevinc Ercan
- Department of Biology and Center for Genomics and Systems Biology, New York UniversityNew YorkUnited States
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19
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Konzman D, Fukushige T, Dagnachew M, Krause M, Hanover JA. O-GlcNAc transferase plays a non-catalytic role in C. elegans male fertility. PLoS Genet 2022; 18:e1010273. [PMID: 36383567 PMCID: PMC9710795 DOI: 10.1371/journal.pgen.1010273] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2022] [Revised: 11/30/2022] [Accepted: 11/08/2022] [Indexed: 11/17/2022] Open
Abstract
Animal behavior is influenced by the competing drives to maintain energy and to reproduce. The balance between these evolutionary pressures and how nutrient signaling pathways intersect with mating remains unclear. The nutrient sensor O-GlcNAc transferase, which post-translationally modifies intracellular proteins with a single monosaccharide, is responsive to cellular nutrient status and regulates diverse biological processes. Though essential in most metazoans, O-GlcNAc transferase (ogt-1) is dispensable in Caenorhabditis elegans, allowing genetic analysis of its physiological roles. Compared to control, ogt-1 males had a four-fold reduction in mean offspring, with nearly two thirds producing zero progeny. Interestingly, we found that ogt-1 males transferred sperm less often, and virgin males had reduced sperm count. ogt-1 males were also less likely to engage in mate-searching and mate-response behaviors. Surprisingly, we found normal fertility for males with hypodermal expression of ogt-1 and for ogt-1 strains with catalytic-dead mutations. This suggests OGT-1 serves a non-catalytic function in the hypodermis impacting male fertility and mating behavior. This study builds upon research on the nutrient sensor O-GlcNAc transferase and demonstrates a role it plays in the interplay between the evolutionary drives for reproduction and survival.
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Affiliation(s)
- Daniel Konzman
- Laboratory of Cellular and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
- Department of Biology, Johns Hopkins University, Baltimore, Maryland, United States of America
| | - Tetsunari Fukushige
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Mesgana Dagnachew
- Laboratory of Cellular and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Michael Krause
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
| | - John A. Hanover
- Laboratory of Cellular and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
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20
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El Mouridi S, Alkhaldi F, Frøkjær-Jensen C. Modular safe-harbor transgene insertion for targeted single-copy and extrachromosomal array integration in Caenorhabditis elegans. G3 (BETHESDA, MD.) 2022; 12:jkac184. [PMID: 35900171 PMCID: PMC9434227 DOI: 10.1093/g3journal/jkac184] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/30/2022] [Accepted: 06/29/2022] [Indexed: 12/02/2022]
Abstract
Efficient and reproducible transgenesis facilitates and accelerates research using genetic model organisms. Here, we describe a modular safe-harbor transgene insertion (MosTI) for use in Caenorhabditis elegans which improves targeted insertion of single-copy transgenes by homology directed repair and targeted integration of extrachromosomal arrays by nonhomologous end-joining. MosTI allows easy conversion between selection markers at insertion site and a collection of universal targeting vectors with commonly used promoters and fluorophores. Insertions are targeted at three permissive safe-harbor intergenic locations and transgenes are reproducibly expressed in somatic and germ cells. Chromosomal integration is mediated by CRISPR/Cas9, and positive selection is based on a set of split markers (unc-119, hygroR, and gfp) where only animals with chromosomal insertions are rescued, resistant to antibiotics, or fluorescent, respectively. Single-copy insertion is efficient using either constitutive or heat-shock inducible Cas9 expression (25-75%) and insertions can be generated from a multiplexed injection mix. Extrachromosomal array integration is also efficient (7-44%) at modular safe-harbor transgene insertion landing sites or at the endogenous unc-119 locus. We use short-read sequencing to estimate the plasmid copy numbers for 8 integrated arrays (6-37 copies) and long-read Nanopore sequencing to determine the structure and size (5.4 Mb) of 1 array. Using universal targeting vectors, standardized insertion strains, and optimized protocols, it is possible to construct complex transgenic strains which should facilitate the study of increasingly complex biological problems in C. elegans.
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Affiliation(s)
- Sonia El Mouridi
- Biological and Environmental Sciences and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia
| | - Faisal Alkhaldi
- Biological and Environmental Sciences and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia
| | - Christian Frøkjær-Jensen
- Biological and Environmental Sciences and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia
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21
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Shah P, Bao Z, Zaidel-Bar R. Visualizing and quantifying molecular and cellular processes in C. elegans using light microscopy. Genetics 2022; 221:6619563. [PMID: 35766819 DOI: 10.1093/genetics/iyac068] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2021] [Accepted: 04/14/2022] [Indexed: 11/14/2022] Open
Abstract
Light microscopes are the cell and developmental biologists' "best friend", providing a means to see structures and follow dynamics from the protein to the organism level. A huge advantage of C. elegans as a model organism is its transparency, which coupled with its small size means that nearly every biological process can be observed and measured with the appropriate probe and light microscope. Continuous improvement in microscope technologies along with novel genome editing techniques to create transgenic probes have facilitated the development and implementation of a dizzying array of methods for imaging worm embryos, larvae and adults. In this review we provide an overview of the molecular and cellular processes that can be visualized in living worms using light microscopy. A partial inventory of fluorescent probes and techniques successfully used in worms to image the dynamics of cells, organelles, DNA, and protein localization and activity is followed by a practical guide to choosing between various imaging modalities, including widefield, confocal, lightsheet, and structured illumination microscopy. Finally, we discuss the available tools and approaches, including machine learning, for quantitative image analysis tasks, such as colocalization, segmentation, object tracking, and lineage tracing. Hopefully, this review will inspire worm researchers who have not yet imaged their worms to begin, and push those who are imaging to go faster, finer, and longer.
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Affiliation(s)
- Pavak Shah
- Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles, Los Angeles 90095, USA
| | - Zhirong Bao
- Developmental Biology Program, Sloan Kettering Institute, New York, New York 10065, USA
| | - Ronen Zaidel-Bar
- Department of Cell and Developmental Biology, Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
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22
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Li X, Li J, Zhu D, Zhang N, Hao X, Zhang W, Zhang Q, Liu Y, Wu X, Tian Y. Protein disulfide isomerase PDI-6 regulates Wnt secretion to coordinate inter-tissue UPR mt activation and lifespan extension in C. elegans. Cell Rep 2022; 39:110931. [PMID: 35675782 DOI: 10.1016/j.celrep.2022.110931] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2021] [Revised: 03/02/2022] [Accepted: 05/18/2022] [Indexed: 11/15/2022] Open
Abstract
Coordination of inter-tissue stress signaling is essential for organismal fitness. Neuronal mitochondrial perturbations activate the mitochondrial unfolded-protein response (UPRmt) in the intestine via the mitokine Wnt signaling in Caenorhabditis elegans. Here, we found that the protein disulfide isomerase PDI-6 coordinates inter-tissue UPRmt signaling via regulating the Wnt ligand EGL-20. PDI-6 is expressed in the endoplasmic reticulum (ER) and interacts with EGL-20 through disulfide bonds that are essential for EGL-20 stability and secretion. pdi-6 deficiency results in misfolded EGL-20, which leads to its degradation via ER-associated protein degradation (ERAD) machinery. Expression of PDI-6 declines drastically with aging, and animals with pdi-6 deficiency have decreased lifespan. Overexpression of PDI-6 is sufficient to maintain Wnt/EGL-20 protein levels during aging, activating the UPRmt, and significantly extending lifespan in a Wnt- and UPRmt-dependent manner. Our study reveals that protein disulfide isomerase facilitates Wnt secretion to coordinate the inter-tissue UPRmt signaling and organismal aging.
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Affiliation(s)
- Xinyu Li
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100093, China
| | - Jiasheng Li
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100093, China
| | - Di Zhu
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100093, China
| | - Ning Zhang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100093, China
| | - Xusheng Hao
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100093, China
| | - Wenfeng Zhang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100093, China
| | - Qian Zhang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
| | - Yangli Liu
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
| | - Xueying Wu
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
| | - Ye Tian
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100093, China; Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming 650223, China.
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23
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Choudhary S, Kashyap SS, Martin RJ, Robertson AP. Advances in our understanding of nematode ion channels as potential anthelmintic targets. Int J Parasitol Drugs Drug Resist 2022; 18:52-86. [PMID: 35149380 PMCID: PMC8841521 DOI: 10.1016/j.ijpddr.2021.12.001] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Revised: 12/14/2021] [Accepted: 12/15/2021] [Indexed: 12/15/2022]
Abstract
Ion channels are specialized multimeric proteins that underlie cell excitability. These channels integrate with a variety of neuromuscular and biological functions. In nematodes, the physiological behaviors including locomotion, navigation, feeding and reproduction, are regulated by these protein entities. Majority of the antinematodal chemotherapeutics target the ion channels to disrupt essential biological functions. Here, we have summarized current advances in our understanding of nematode ion channel pharmacology. We review cys-loop ligand gated ion channels (LGICs), including nicotinic acetylcholine receptors (nAChRs), acetylcholine-chloride gated ion channels (ACCs), glutamate-gated chloride channels (GluCls), and GABA (γ-aminobutyric acid) receptors, and other ionotropic receptors (transient receptor potential (TRP) channels and potassium ion channels). We have provided an update on the pharmacological properties of these channels from various nematodes. This article catalogs the differences in ion channel composition and resulting pharmacology in the phylum Nematoda. This diversity in ion channel subunit repertoire and pharmacology emphasizes the importance of pursuing species-specific drug target research. In this review, we have provided an overview of recent advances in techniques and functional assays available for screening ion channel properties and their application.
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Affiliation(s)
- Shivani Choudhary
- Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
| | - Sudhanva S Kashyap
- Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
| | - Richard J Martin
- Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
| | - Alan P Robertson
- Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA.
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24
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Castiglioni VG, Ramalho JJ, Kroll JR, Stucchi R, van Beuzekom H, Schmidt R, Altelaar M, Boxem M. Identification and characterization of Crumbs polarity complex proteins in Caenorhabditis elegans. J Biol Chem 2022; 298:101786. [PMID: 35247383 PMCID: PMC9006659 DOI: 10.1016/j.jbc.2022.101786] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2021] [Revised: 02/12/2022] [Accepted: 02/16/2022] [Indexed: 11/23/2022] Open
Abstract
Crumbs proteins are evolutionarily conserved transmembrane proteins with essential roles in promoting the formation of the apical domain in epithelial cells. The short intracellular tail of Crumbs proteins are known to interact with several proteins, including the scaffolding protein PALS1 (protein associated with LIN7, Stardust in Drosophila). PALS1 in turn binds to a second scaffolding protein PATJ (PALS1-associated tight junction protein) to form the core Crumbs/PALS1/PATJ complex. While essential roles in epithelial organization have been shown for Crumbs proteins in Drosophila and mammalian systems, the three Caenorhabditis elegans crumbs genes are dispensable for epithelial polarization and development. Here, we investigated the presence and function of PALS1 and PATJ orthologs in C. elegans. We identified MAGU-2 as the C. elegans ortholog of PALS1 and show that MAGU-2 interacts with all three Crumbs proteins and localizes to the apical membrane domain of intestinal epithelial cells in a Crumbs-dependent fashion. Similar to crumbs mutants, magu-2 deletion showed no epithelial polarity defects. We also identified MPZ-1 as a candidate ortholog of PATJ based on the physical interaction with MAGU-2 and sequence similarity with PATJ proteins. However, MPZ-1 is not broadly expressed in epithelial tissues and, therefore, not likely a core component of the C. elegans Crumbs complex. Finally, we show overexpression of the Crumbs proteins EAT-20 or CRB-3 can lead to apical membrane expansion in the intestine. Our results shed light on the composition of the C. elegans Crumbs complex and indicate that the role of Crumbs proteins in promoting apical domain formation is conserved.
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Affiliation(s)
- Victoria G Castiglioni
- Division of Developmental Biology, Department of Biology, Faculty of Science, Institute of Biodynamics and Biocomplexity, Utrecht University, Utrecht, The Netherlands
| | - João J Ramalho
- Division of Developmental Biology, Department of Biology, Faculty of Science, Institute of Biodynamics and Biocomplexity, Utrecht University, Utrecht, The Netherlands
| | - Jason R Kroll
- Division of Developmental Biology, Department of Biology, Faculty of Science, Institute of Biodynamics and Biocomplexity, Utrecht University, Utrecht, The Netherlands
| | - Riccardo Stucchi
- Division of Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Institute of Biodynamics and Biocomplexity, Utrecht University, Utrecht, The Netherlands; Division of Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands
| | - Hanna van Beuzekom
- Division of Developmental Biology, Department of Biology, Faculty of Science, Institute of Biodynamics and Biocomplexity, Utrecht University, Utrecht, The Netherlands
| | - Ruben Schmidt
- Division of Developmental Biology, Department of Biology, Faculty of Science, Institute of Biodynamics and Biocomplexity, Utrecht University, Utrecht, The Netherlands
| | - Maarten Altelaar
- Division of Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands
| | - Mike Boxem
- Division of Developmental Biology, Department of Biology, Faculty of Science, Institute of Biodynamics and Biocomplexity, Utrecht University, Utrecht, The Netherlands.
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25
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Blomme J, Develtere W, Köse A, Arraiza Ribera J, Brugmans C, Jaraba-Wallace J, Decaestecker W, Rombaut D, Baekelandt A, Daniel Fernández Fernández Á, Van Breusegem F, Inzé D, Jacobs T. The heat is on: a simple method to increase genome editing efficiency in plants. BMC PLANT BIOLOGY 2022; 22:142. [PMID: 35331142 PMCID: PMC8951696 DOI: 10.1186/s12870-022-03519-7] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/24/2021] [Accepted: 03/08/2022] [Indexed: 05/30/2023]
Abstract
BACKGROUND Precision genome mutagenesis using CRISPR/Cas has become the standard method to generate mutant plant lines. Several improvements have been made to increase mutagenesis efficiency, either through vector optimisation or the application of heat stress. RESULTS Here, we present a simplified heat stress assay that can be completed in six days using commonly-available laboratory equipment. We show that three heat shocks (3xHS) efficiently increases indel efficiency of LbCas12a and Cas9, irrespective of the target sequence or the promoter used to express the nuclease. The generated indels are primarily somatic, but for three out of five targets we demonstrate that up to 25% more biallelic mutations are transmitted to the progeny when heat is applied compared to non-heat controls. We also applied our heat treatment to lines containing CRISPR base editors and observed a 22-27% increase in the percentage of C-to-T base editing. Furthermore, we test the effect of 3xHS on generating large deletions and a homologous recombination reporter. Interestingly, we observed no positive effect of 3xHS treatment on either approach using our conditions. CONCLUSIONS Together, our experiments show that heat treatment is consistently effective at increasing the number of somatic mutations using many CRISPR approaches in plants and in some cases can increase the recovery of mutant progeny.
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Affiliation(s)
- Jonas Blomme
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium
- VIB Center for Plant Systems Biology, 9052, Ghent, Belgium
- Phycology Research Group, Department of Biology, Ghent University, 9000, Ghent, Belgium
| | - Ward Develtere
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium
- VIB Center for Plant Systems Biology, 9052, Ghent, Belgium
| | - Ayse Köse
- Bioengineering Department, Ege University, 35100, Izmir, Turkey
| | - Júlia Arraiza Ribera
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium
- VIB Center for Plant Systems Biology, 9052, Ghent, Belgium
| | - Christophe Brugmans
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium
- VIB Center for Plant Systems Biology, 9052, Ghent, Belgium
| | - Jessica Jaraba-Wallace
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium
- VIB Center for Plant Systems Biology, 9052, Ghent, Belgium
| | - Ward Decaestecker
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium
- VIB Center for Plant Systems Biology, 9052, Ghent, Belgium
| | - Debbie Rombaut
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium
- VIB Center for Plant Systems Biology, 9052, Ghent, Belgium
| | - Alexandra Baekelandt
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium
- VIB Center for Plant Systems Biology, 9052, Ghent, Belgium
| | - Álvaro Daniel Fernández Fernández
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium
- VIB Center for Plant Systems Biology, 9052, Ghent, Belgium
| | - Frank Van Breusegem
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium
- VIB Center for Plant Systems Biology, 9052, Ghent, Belgium
| | - Dirk Inzé
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium
- VIB Center for Plant Systems Biology, 9052, Ghent, Belgium
| | - Thomas Jacobs
- Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium.
- VIB Center for Plant Systems Biology, 9052, Ghent, Belgium.
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26
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Stundl J, Soukup V, Franěk R, Pospisilova A, Psutkova V, Pšenička M, Cerny R, Bronner ME, Medeiros DM, Jandzik D. Efficient CRISPR Mutagenesis in Sturgeon Demonstrates Its Utility in Large, Slow-Maturing Vertebrates. Front Cell Dev Biol 2022; 10:750833. [PMID: 35223827 PMCID: PMC8867083 DOI: 10.3389/fcell.2022.750833] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2021] [Accepted: 01/17/2022] [Indexed: 11/14/2022] Open
Abstract
In the last decade, the CRISPR/Cas9 bacterial virus defense system has been adapted as a user-friendly, efficient, and precise method for targeted mutagenesis in eukaryotes. Though CRISPR/Cas9 has proven effective in a diverse range of organisms, it is still most often used to create mutant lines in lab-reared genetic model systems. However, one major advantage of CRISPR/Cas9 mutagenesis over previous gene targeting approaches is that its high efficiency allows the immediate generation of near-null mosaic mutants. This feature could potentially allow genotype to be linked to phenotype in organisms with life histories that preclude the establishment of purebred genetic lines; a group that includes the vast majority of vertebrate species. Of particular interest to scholars of early vertebrate evolution are several long-lived and slow-maturing fishes that diverged from two dominant modern lineages, teleosts and tetrapods, in the Ordovician, or before. These early-diverging or "basal" vertebrates include the jawless cyclostomes, cartilaginous fishes, and various non-teleost ray-finned fishes. In addition to occupying critical phylogenetic positions, these groups possess combinations of derived and ancestral features not seen in conventional model vertebrates, and thus provide an opportunity for understanding the genetic bases of such traits. Here we report successful use of CRISPR/Cas9 mutagenesis in one such non-teleost fish, sterlet Acipenser ruthenus, a small species of sturgeon. We introduced mutations into the genes Tyrosinase, which is needed for melanin production, and Sonic hedgehog, a pleiotropic developmental regulator with diverse roles in early embryonic patterning and organogenesis. We observed disruption of both loci and the production of consistent phenotypes, including both near-null mutants' various hypomorphs. Based on these results, and previous work in lamprey and amphibians, we discuss how CRISPR/Cas9 F0 mutagenesis may be successfully adapted to other long-lived, slow-maturing aquatic vertebrates and identify the ease of obtaining and injecting eggs and/or zygotes as the main challenges.
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Affiliation(s)
- Jan Stundl
- Department of Zoology, Faculty of Science, Charles University, Prague, Czechia
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, United States
- South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Faculty of Fisheries and Protection of Waters, University of South Bohemia in České Budějovice, Vodňany, Czechia
| | - Vladimír Soukup
- Department of Zoology, Faculty of Science, Charles University, Prague, Czechia
| | - Roman Franěk
- South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Faculty of Fisheries and Protection of Waters, University of South Bohemia in České Budějovice, Vodňany, Czechia
| | - Anna Pospisilova
- Department of Zoology, Faculty of Science, Charles University, Prague, Czechia
| | - Viktorie Psutkova
- Department of Zoology, Faculty of Science, Charles University, Prague, Czechia
| | - Martin Pšenička
- South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Faculty of Fisheries and Protection of Waters, University of South Bohemia in České Budějovice, Vodňany, Czechia
| | - Robert Cerny
- Department of Zoology, Faculty of Science, Charles University, Prague, Czechia
| | - Marianne E. Bronner
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, United States
| | - Daniel Meulemans Medeiros
- Department of Ecology and Evolutionary Biology, University of Colorado in Boulder, Boulder, CO, United States
| | - David Jandzik
- Department of Zoology, Faculty of Science, Charles University, Prague, Czechia
- Department of Zoology, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia
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27
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You H, Gordon CA, MacGregor SR, Cai P, McManus DP. Potential of the CRISPR-Cas system for improved parasite diagnosis: CRISPR-Cas mediated diagnosis in parasitic infections: CRISPR-Cas mediated diagnosis in parasitic infections. Bioessays 2022; 44:e2100286. [PMID: 35142378 DOI: 10.1002/bies.202100286] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2021] [Revised: 01/26/2022] [Accepted: 01/27/2022] [Indexed: 12/26/2022]
Abstract
CRISPR-Cas technology accelerates development of fast, accurate, and portable diagnostic tools, typified by recent applications in COVID-19 diagnosis. Parasitic helminths cause devastating diseases afflicting 1.5 billion people globally, representing a significant public health and economic burden, especially in developing countries. Currently available diagnostic tests for worm infection are neither sufficiently sensitive nor field-friendly for use in low-endemic or resource-poor settings, leading to underestimation of true prevalence rates. Mass drug administration programs are unsustainable long-term, and diagnostic tools - required to be rapid, specific, sensitive, cost-effective, and user-friendly without specialized equipment and expertise - are urgently needed for rapid mapping of helminthic diseases and monitoring control programs. We describe the key features of the CRISPR-Cas12/13 system and emphasise its potential for the development of effective tools for the diagnosis of parasitic and other neglected tropical diseases (NTDs), a key recommendation of the NTDs 2021-2030 roadmap released by the World Health Organization.
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Affiliation(s)
- Hong You
- Immunology Department, QIMR Berghofer Medical Research Institute, Queensland, Australia
| | - Catherine A Gordon
- Immunology Department, QIMR Berghofer Medical Research Institute, Queensland, Australia
| | - Skye R MacGregor
- Immunology Department, QIMR Berghofer Medical Research Institute, Queensland, Australia
| | - Pengfei Cai
- Immunology Department, QIMR Berghofer Medical Research Institute, Queensland, Australia
| | - Donald P McManus
- Immunology Department, QIMR Berghofer Medical Research Institute, Queensland, Australia
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28
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Hiraga H, Ishita Y, Chihara T, Okumura M. Efficient visual screening of CRISPR/Cas9 genome editing in the nematode Pristionchus pacificus. Dev Growth Differ 2021; 63:488-500. [PMID: 34813661 DOI: 10.1111/dgd.12761] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2021] [Revised: 09/17/2021] [Accepted: 09/30/2021] [Indexed: 12/22/2022]
Abstract
CRISPR/Cas9 genome editing has been applied to a wide variety of organisms, including nematodes such as Caenorhabditis elegans and Pristionchus pacificus. In these nematodes, genome editing is achieved by microinjection of Cas9 protein and guide RNA into the hermaphrodite gonads. However, P. pacificus is less efficient in CRISPR/Cas9 genome editing and exogenous gene expression. Therefore, it takes considerable time and effort to screen for target mutants if there are no visual markers that indicate successful injection. To overcome this problem, co-injection markers (gRNA for Ppa-prl-1, which induces the roller phenotype, and Ppa-egl-20p::turboRFP, a plasmid expressing a fluorescent protein) have been developed in P. pacificus. By selecting worms with the roller phenotype or turboRFP expression, screening efficiency is substantially increased to obtain worms with desired mutations. Here, we describe a step-by-step protocol for the visual screening system for CRISPR/Cas9 genome editing in P. pacificus. We also describe technical tips for microinjection, which is difficult for beginners. This protocol will facilitate genome editing in P. pacificus and may be applied to other nematode species.
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Affiliation(s)
- Hirokuni Hiraga
- Program of Biomedical Science, Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima, Japan
| | - Yuuki Ishita
- Program of Biomedical Science, Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima, Japan
| | - Takahiro Chihara
- Program of Biomedical Science, Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima, Japan.,Program of Basic Biology, Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima, Japan
| | - Misako Okumura
- Program of Biomedical Science, Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima, Japan.,Program of Basic Biology, Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima, Japan
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29
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Schwartz ML, Davis MW, Rich MS, Jorgensen EM. High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome. PLoS Genet 2021; 17:e1009755. [PMID: 34748534 PMCID: PMC8601624 DOI: 10.1371/journal.pgen.1009755] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2021] [Revised: 11/18/2021] [Accepted: 10/16/2021] [Indexed: 11/24/2022] Open
Abstract
Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple, short homology arm GFP insertions, 50-100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits.
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Affiliation(s)
- Matthew L. Schwartz
- Howard Hughes Medical Institute and School of Biological Sciences, University of Utah, Salt Lake City, Utah, United States of America
| | - M. Wayne Davis
- Howard Hughes Medical Institute and School of Biological Sciences, University of Utah, Salt Lake City, Utah, United States of America
| | - Matthew S. Rich
- Howard Hughes Medical Institute and School of Biological Sciences, University of Utah, Salt Lake City, Utah, United States of America
| | - Erik M. Jorgensen
- Howard Hughes Medical Institute and School of Biological Sciences, University of Utah, Salt Lake City, Utah, United States of America
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30
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Huang G, de Jesus B, Koh A, Blanco S, Rettmann A, DeMott E, Sylvester M, Ren C, Meng C, Waterland S, Rhodes A, Alicea P, Flynn A, Dickinson DJ, Doonan R. Improved CRISPR/Cas9 knock-in efficiency via the self-excising cassette (SEC) selection method in C. elegans. MICROPUBLICATION BIOLOGY 2021; 2021. [PMID: 34549176 PMCID: PMC8449260 DOI: 10.17912/micropub.biology.000460] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/01/2021] [Revised: 09/12/2021] [Accepted: 09/13/2021] [Indexed: 11/08/2022]
Abstract
Streamlined, selection-based CRISPR knock-in protocols for C. elegans were first introduced six years ago (Dickinson et al. 2015; Schwartz and Jorgensen 2016). Though these selection-based approaches are powerful, one drawback has been the requirement to inject large numbers of P0 worms (~30-60 per gene target). We have found that a combination of high-purity DNA and a lower concentration of Cas9/sgRNA plasmid dramatically improves efficiency, often resulting in multiple independent CRISPR knock-ins via as few as 10 injected worms, comparable to the efficiency of melted dsDNA templates and purified Cas9 protein (Dokshin et al. 2018; Ghanta and Mello 2020).
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Affiliation(s)
- George Huang
- Glow Worms Stream, Freshman Research Initiative, Texas Institute for Discovery Education in Science, College of Natural Sciences, The University of Texas at Austin, Austin TX, USA
| | - Bailey de Jesus
- Glow Worms Stream, Freshman Research Initiative, Texas Institute for Discovery Education in Science, College of Natural Sciences, The University of Texas at Austin, Austin TX, USA
| | - Alex Koh
- Department of Molecular Biosciences, The University of Texas at Austin, Austin TX, USA
| | - Sara Blanco
- Glow Worms Stream, Freshman Research Initiative, Texas Institute for Discovery Education in Science, College of Natural Sciences, The University of Texas at Austin, Austin TX, USA
| | - Aubrie Rettmann
- Glow Worms Stream, Freshman Research Initiative, Texas Institute for Discovery Education in Science, College of Natural Sciences, The University of Texas at Austin, Austin TX, USA
| | - Ella DeMott
- Glow Worms Stream, Freshman Research Initiative, Texas Institute for Discovery Education in Science, College of Natural Sciences, The University of Texas at Austin, Austin TX, USA
| | - Melynda Sylvester
- Glow Worms Stream, Freshman Research Initiative, Texas Institute for Discovery Education in Science, College of Natural Sciences, The University of Texas at Austin, Austin TX, USA
| | - Cassie Ren
- Glow Worms Stream, Freshman Research Initiative, Texas Institute for Discovery Education in Science, College of Natural Sciences, The University of Texas at Austin, Austin TX, USA
| | - Carrie Meng
- Glow Worms Stream, Freshman Research Initiative, Texas Institute for Discovery Education in Science, College of Natural Sciences, The University of Texas at Austin, Austin TX, USA
| | - Skye Waterland
- Glow Worms Stream, Freshman Research Initiative, Texas Institute for Discovery Education in Science, College of Natural Sciences, The University of Texas at Austin, Austin TX, USA
| | - Anita Rhodes
- Glow Worms Stream, Freshman Research Initiative, Texas Institute for Discovery Education in Science, College of Natural Sciences, The University of Texas at Austin, Austin TX, USA
| | - Persephone Alicea
- Glow Worms Stream, Freshman Research Initiative, Texas Institute for Discovery Education in Science, College of Natural Sciences, The University of Texas at Austin, Austin TX, USA
| | - Abbey Flynn
- Glow Worms Stream, Freshman Research Initiative, Texas Institute for Discovery Education in Science, College of Natural Sciences, The University of Texas at Austin, Austin TX, USA
| | - Daniel J Dickinson
- Department of Molecular Biosciences, The University of Texas at Austin, Austin TX, USA
| | - Ryan Doonan
- Glow Worms Stream, Freshman Research Initiative, Texas Institute for Discovery Education in Science, College of Natural Sciences, The University of Texas at Austin, Austin TX, USA.,Department of Molecular Biosciences, The University of Texas at Austin, Austin TX, USA
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31
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Hwang HY, Wang J. Fast genetic mapping using insertion-deletion polymorphisms in Caenorhabditis elegans. Sci Rep 2021; 11:11017. [PMID: 34040027 PMCID: PMC8155061 DOI: 10.1038/s41598-021-90190-x] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2020] [Accepted: 05/07/2021] [Indexed: 11/09/2022] Open
Abstract
Genetic mapping is used in forward genetics to narrow the list of candidate mutations and genes corresponding to the mutant phenotype of interest. Even with modern advances in biology such as efficient identification of candidate mutations by whole-genome sequencing, mapping remains critical in pinpointing the responsible mutation. Here we describe a simple, fast, and affordable mapping toolkit that is particularly suitable for mapping in Caenorhabditis elegans. This mapping method uses insertion-deletion polymorphisms or indels that could be easily detected instead of single nucleotide polymorphisms in commonly used Hawaiian CB4856 mapping strain. The materials and methods were optimized so that mapping could be performed using tiny amount of genetic material without growing many large populations of mutants for DNA purification. We performed mapping of previously known and unknown mutations to show strengths and weaknesses of this method and to present examples of completed mapping. For situations where Hawaiian CB4856 is unsuitable, we provide an annotated list of indels as a basis for fast and easy mapping using other wild isolates. Finally, we provide rationale for using this mapping method over other alternatives as a part of a comprehensive strategy also involving whole-genome sequencing and other methods.
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Affiliation(s)
- Ho-Yon Hwang
- Department of Biochemistry and Molecular Biology, Department of Neuroscience, Johns Hopkins University, 615 N. Wolfe Street, E8410, Baltimore, MD, 21205, USA
| | - Jiou Wang
- Department of Biochemistry and Molecular Biology, Department of Neuroscience, Johns Hopkins University, 615 N. Wolfe Street, E8410, Baltimore, MD, 21205, USA.
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32
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Froehlich JJ, Uyar B, Herzog M, Theil K, Glažar P, Akalin A, Rajewsky N. Parallel genetics of regulatory sequences using scalable genome editing in vivo. Cell Rep 2021; 35:108988. [PMID: 33852857 DOI: 10.1016/j.celrep.2021.108988] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2020] [Revised: 01/13/2021] [Accepted: 03/23/2021] [Indexed: 12/27/2022] Open
Abstract
How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. Regulatory elements must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3' UTRs of 16 genes in C. elegans. Our software crispr-DART analyzes indel mutations in targeted DNA sequencing. We quantify the impact of mutations on expression and fitness by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3' UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression independently of each other. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of regulatory sequences directly in animals.
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Affiliation(s)
- Jonathan J Froehlich
- Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, Germany
| | - Bora Uyar
- Bioinformatics and Omics Data Science Platform, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, Germany
| | - Margareta Herzog
- Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, Germany
| | - Kathrin Theil
- Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, Germany
| | - Petar Glažar
- Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, Germany
| | - Altuna Akalin
- Bioinformatics and Omics Data Science Platform, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, Germany
| | - Nikolaus Rajewsky
- Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, Germany.
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33
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Vicencio J, Cerón J. A Living Organism in your CRISPR Toolbox: Caenorhabditis elegans Is a Rapid and Efficient Model for Developing CRISPR-Cas Technologies. CRISPR J 2021; 4:32-42. [PMID: 33538637 DOI: 10.1089/crispr.2020.0103] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The Cas9 nuclease from Streptococcus pyogenes (SpCas9) is the most popular enzyme for CRISPR technologies. However, considering the wide diversity of microorganisms (discovered and still unknown), a massive number of CRISPR effectors are being and will be identified and characterized in the search of optimal Cas variants for each of the many applications of CRISPR. In this context, a versatile and efficient multicellular system for CRISPR editing such as Caenorhabditis elegans would be of great help in the development of these effectors. Here, we highlight the benefits of using C. elegans for the rapid evaluation of new CRISPR effectors, and for optimizing CRISPR efficiency in animals in several ways such as by modulating the balance between repair pathways, modifying chromatin accessibility, or controlling the expression and activity of nucleases and guide RNAs.
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Affiliation(s)
- Jeremy Vicencio
- Modeling human diseases in C. elegans Group, Genes, Disease and Therapy Program, Bellvitge Biomedical Research Institute-IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain
| | - Julián Cerón
- Modeling human diseases in C. elegans Group, Genes, Disease and Therapy Program, Bellvitge Biomedical Research Institute-IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain
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34
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Du X, McManus DP, French JD, Jones MK, You H. CRISPR/Cas9: A new tool for the study and control of helminth parasites. Bioessays 2020; 43:e2000185. [PMID: 33145822 DOI: 10.1002/bies.202000185] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2020] [Revised: 09/16/2020] [Accepted: 09/18/2020] [Indexed: 12/17/2022]
Abstract
Recent reports of CRISPR/Cas9 genome editing in parasitic helminths open up new avenues for research on these dangerous pathogens. However, the complex morphology and life cycles inherent to these parasites present obstacles for the efficient application of CRISPR/Cas9-targeted mutagenesis. This is especially true with the trematode flukes where only modest levels of gene mutation efficiency have been achieved. Current major challenges in the application of CRISPR/Cas9 for study of parasitic worms thus lie in enhancing gene mutation efficiency and overcoming issues involved in host passage so that mutated parasites survive. Strategies developed for CRISPR/Cas9 studies on Caenorhabditis elegans, protozoa and mammalian cells, including novel delivery methods, the choice of selectable markers, and refining mutation precision represent novel tactics whereby these impediments can be overcome. Furthermore, employing CRISPR/Cas9-mediated gene drive to interfere with vector transmission represents a novel approach for the control of parasitic worms that is worthy of further exploration.
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Affiliation(s)
- Xiaofeng Du
- Immunology Department, QIMR Berghofer Medical Research Institute, Herston, Brisbane, Queensland, Australia.,Faculty of Medicine, The University of Queensland, Herston, Brisbane, Queensland, Australia
| | - Donald P McManus
- Immunology Department, QIMR Berghofer Medical Research Institute, Herston, Brisbane, Queensland, Australia.,Faculty of Medicine, The University of Queensland, Herston, Brisbane, Queensland, Australia
| | - Juliet D French
- Genetics & Computational Biology Department, QIMR Berghofer Medical Research Institute, Herston, Brisbane, Queensland, Australia
| | - Malcolm K Jones
- School of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia
| | - Hong You
- Immunology Department, QIMR Berghofer Medical Research Institute, Herston, Brisbane, Queensland, Australia
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35
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McDiarmid TA, Au V, Moerman DG, Rankin CH. Peel-1 negative selection promotes screening-free CRISPR-Cas9 genome editing in Caenorhabditis elegans. PLoS One 2020; 15:e0238950. [PMID: 32960926 PMCID: PMC7508457 DOI: 10.1371/journal.pone.0238950] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2020] [Accepted: 08/26/2020] [Indexed: 02/07/2023] Open
Abstract
Improved genome engineering methods that enable automation of large and precise edits are essential for systematic investigations of genome function. We adapted peel-1 negative selection to an optimized Dual-Marker Selection (DMS) cassette protocol for CRISPR-Cas9 genome engineering in Caenorhabditis elegans and observed robust increases in multiple measures of efficiency that were consistent across injectors and four genomic loci. The use of Peel-1-DMS selection killed animals harboring transgenes as extrachromosomal arrays and spared genome-edited integrants, often circumventing the need for visual screening to identify genome-edited animals. To demonstrate the applicability of the approach, we created deletion alleles in the putative proteasomal subunit pbs-1 and the uncharacterized gene K04F10.3 and used machine vision to automatically characterize their phenotypic profiles, revealing homozygous essential and heterozygous behavioral phenotypes. These results provide a robust and scalable approach to rapidly generate and phenotype genome-edited animals without the need for screening or scoring by eye.
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Affiliation(s)
- Troy A. McDiarmid
- Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, British Columbia, Canada
| | - Vinci Au
- Department of Zoology, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada
| | - Donald G. Moerman
- Department of Zoology, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada
| | - Catharine H. Rankin
- Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, British Columbia, Canada
- Department of Psychology, University of British Columbia, Vancouver, British Columbia, Canada
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36
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Ramalho JJ, Sepers JJ, Nicolle O, Schmidt R, Cravo J, Michaux G, Boxem M. C-terminal phosphorylation modulates ERM-1 localization and dynamics to control cortical actin organization and support lumen formation during Caenorhabditiselegans development. Development 2020; 147:dev188011. [PMID: 32586975 PMCID: PMC10755404 DOI: 10.1242/dev.188011] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2020] [Accepted: 06/13/2020] [Indexed: 12/31/2023]
Abstract
ERM proteins are conserved regulators of cortical membrane specialization that function as membrane-actin linkers and molecular hubs. The activity of ERM proteins requires a conformational switch from an inactive cytoplasmic form into an active membrane- and actin-bound form, which is thought to be mediated by sequential PIP2 binding and phosphorylation of a conserved C-terminal threonine residue. Here, we use the single Caenorhabditiselegans ERM ortholog, ERM-1, to study the contribution of these regulatory events to ERM activity and tissue formation in vivo Using CRISPR/Cas9-generated erm-1 mutant alleles, we demonstrate that a PIP2-binding site is crucially required for ERM-1 function. By contrast, dynamic regulation of C-terminal T544 phosphorylation is not essential but modulates ERM-1 apical localization and dynamics in a tissue-specific manner, to control cortical actin organization and support lumen formation in epithelial tubes. Our work highlights the dynamic nature of ERM protein regulation during tissue morphogenesis and the importance of C-terminal phosphorylation in fine-tuning ERM activity in a tissue-specific context.
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Affiliation(s)
- João J Ramalho
- Division of Developmental Biology, Institute of Biodynamics and Biocomplexity, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands
| | - Jorian J Sepers
- Division of Developmental Biology, Institute of Biodynamics and Biocomplexity, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands
| | - Ophélie Nicolle
- Univ Rennes, CNRS, IGDR (Institut de Génétique et de Développement de Rennes), UMR 6290, F-35000 Rennes, France
| | - Ruben Schmidt
- Division of Developmental Biology, Institute of Biodynamics and Biocomplexity, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands
| | - Janine Cravo
- Division of Developmental Biology, Institute of Biodynamics and Biocomplexity, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands
| | - Grégoire Michaux
- Univ Rennes, CNRS, IGDR (Institut de Génétique et de Développement de Rennes), UMR 6290, F-35000 Rennes, France
| | - Mike Boxem
- Division of Developmental Biology, Institute of Biodynamics and Biocomplexity, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands
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37
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Breunig CT, Köferle A, Neuner AM, Wiesbeck MF, Baumann V, Stricker SH. CRISPR Tools for Physiology and Cell State Changes: Potential of Transcriptional Engineering and Epigenome Editing. Physiol Rev 2020; 101:177-211. [PMID: 32525760 DOI: 10.1152/physrev.00034.2019] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Given the large amount of genome-wide data that have been collected during the last decades, a good understanding of how and why cells change during development, homeostasis, and disease might be expected. Unfortunately, the opposite is true; triggers that cause cellular state changes remain elusive, and the underlying molecular mechanisms are poorly understood. Although genes with the potential to influence cell states are known, the historic dependency on methods that manipulate gene expression outside the endogenous chromatin context has prevented us from understanding how cells organize, interpret, and protect cellular programs. Fortunately, recent methodological innovations are now providing options to answer these outstanding questions, by allowing to target and manipulate individual genomic and epigenomic loci. In particular, three experimental approaches are now feasible due to DNA targeting tools, namely, activation and/or repression of master transcription factors in their endogenous chromatin context; targeting transcription factors to endogenous, alternative, or inaccessible sites; and finally, functional manipulation of the chromatin context. In this article, we discuss the molecular basis of DNA targeting tools and review the potential of these new technologies before we summarize how these have already been used for the manipulation of cellular states and hypothesize about future applications.
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Affiliation(s)
- Christopher T Breunig
- MCN Junior Research Group, Munich Center for Neurosciences, Ludwig-Maximilian- Universität, BioMedical Center, Planegg-Martinsried, Germany; and Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, BioMedical Center, Planegg-Martinsried, Germany
| | - Anna Köferle
- MCN Junior Research Group, Munich Center for Neurosciences, Ludwig-Maximilian- Universität, BioMedical Center, Planegg-Martinsried, Germany; and Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, BioMedical Center, Planegg-Martinsried, Germany
| | - Andrea M Neuner
- MCN Junior Research Group, Munich Center for Neurosciences, Ludwig-Maximilian- Universität, BioMedical Center, Planegg-Martinsried, Germany; and Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, BioMedical Center, Planegg-Martinsried, Germany
| | - Maximilian F Wiesbeck
- MCN Junior Research Group, Munich Center for Neurosciences, Ludwig-Maximilian- Universität, BioMedical Center, Planegg-Martinsried, Germany; and Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, BioMedical Center, Planegg-Martinsried, Germany
| | - Valentin Baumann
- MCN Junior Research Group, Munich Center for Neurosciences, Ludwig-Maximilian- Universität, BioMedical Center, Planegg-Martinsried, Germany; and Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, BioMedical Center, Planegg-Martinsried, Germany
| | - Stefan H Stricker
- MCN Junior Research Group, Munich Center for Neurosciences, Ludwig-Maximilian- Universität, BioMedical Center, Planegg-Martinsried, Germany; and Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, BioMedical Center, Planegg-Martinsried, Germany
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38
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Culp E, Richman C, Sharanya D, Jhaveri N, van den Berg W, Gupta BP. Genome editing in the nematode Caenorhabditis briggsae using the CRISPR/Cas9 system. Biol Methods Protoc 2020; 5:bpaa003. [PMID: 32395632 PMCID: PMC7200835 DOI: 10.1093/biomethods/bpaa003] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2019] [Revised: 01/27/2020] [Accepted: 02/07/2020] [Indexed: 12/26/2022] Open
Abstract
The CRISPR/Cas system has recently emerged as a powerful tool to engineer the genome of an organism. The system is adopted from bacteria where it confers immunity against invading foreign DNA. This work reports the first successful use of the CRISPR/Cas system in Caenorhabditis briggsae (a cousin of the well-known nematode C. elegans), to generate mutations via non-homologous end joining. We recovered deletion alleles of several conserved genes by microinjecting plasmids that express Cas9 endonuclease and an engineered CRISPR RNA corresponding to the DNA sequence to be cleaved. Evidence for somatic mutations and off-target mutations are also reported. Our approach allows for the generation of loss-of-function mutations in C. briggsae genes thereby facilitating a comparative study of gene function.
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Affiliation(s)
- Elizabeth Culp
- Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S-4K1, Canada
| | - Cory Richman
- Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S-4K1, Canada
| | - Devika Sharanya
- Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S-4K1, Canada
| | - Nikita Jhaveri
- Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S-4K1, Canada
| | - Wouter van den Berg
- Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S-4K1, Canada
| | - Bhagwati P Gupta
- Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S-4K1, Canada
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39
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van Bostelen I, van Schendel R, Romeijn R, Tijsterman M. Translesion synthesis polymerases are dispensable for C. elegans reproduction but suppress genome scarring by polymerase theta-mediated end joining. PLoS Genet 2020; 16:e1008759. [PMID: 32330130 PMCID: PMC7202663 DOI: 10.1371/journal.pgen.1008759] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2020] [Revised: 05/06/2020] [Accepted: 04/06/2020] [Indexed: 12/30/2022] Open
Abstract
Bases within DNA are frequently damaged, producing obstacles to efficient and accurate DNA replication by replicative polymerases. Translesion synthesis (TLS) polymerases, via their ability to catalyze nucleotide additions to growing DNA chains across DNA lesions, promote replication of damaged DNA, thus preventing checkpoint activation, genome instability and cell death. In this study, we used C. elegans to determine the contribution of TLS activity on long-term stability of an animal genome. We monitored and compared the types of mutations that accumulate in REV1, REV3, POLH1 and POLK deficient animals that were grown under unchallenged conditions. We also addressed redundancies in TLS activity by combining all deficiencies. Remarkably, animals that are deficient for all Y-family polymerases as well as animals that have lost all TLS activity are viable and produce progeny, demonstrating that TLS is not essential for animal life. Whole genome sequencing analyses, however, reveal that TLS is needed to prevent genomic scars from accumulating. These scars, which are the product of polymerase theta-mediated end joining (TMEJ), are found overrepresented at guanine bases, consistent with TLS suppressing DNA double-strand breaks (DSBs) from occurring at replication-blocking guanine adducts. We found that in C. elegans, TLS across spontaneous damage is predominantly error free and anti-clastogenic, and thus ensures preservation of genetic information.
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Affiliation(s)
- Ivo van Bostelen
- Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
| | - Robin van Schendel
- Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
| | - Ron Romeijn
- Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
| | - Marcel Tijsterman
- Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
- Institute of Biology Leiden, Leiden University, Leiden, The Netherlands
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40
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Haroon S, Li A, Weinert JL, Fritsch C, Ericson NG, Alexander-Floyd J, Braeckman BP, Haynes CM, Bielas JH, Gidalevitz T, Vermulst M. Multiple Molecular Mechanisms Rescue mtDNA Disease in C. elegans. Cell Rep 2019; 22:3115-3125. [PMID: 29562168 PMCID: PMC6106782 DOI: 10.1016/j.celrep.2018.02.099] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2017] [Revised: 12/07/2017] [Accepted: 02/25/2018] [Indexed: 01/03/2023] Open
Abstract
Genetic instability of the mitochondrial genome (mtDNA) plays an important role in human aging and disease. Thus far, it has proven difficult to develop successful treatment strategies for diseases that are caused by mtDNA instability. To address this issue, we developed a model of mtDNA disease in the nematode C. elegans, an animal model that can rapidly be screened for genes and biological pathways that reduce mitochondrial pathology. These worms recapitulate all the major hallmarks of mtDNA disease in humans, including increased mtDNA instability, loss of respiration, reduced neuromuscular function, and a shortened lifespan. We found that these phenotypes could be rescued by intervening in numerous biological pathways, including IGF-1/insulin signaling, mitophagy, and the mitochondrial unfolded protein response, suggesting that it may be possible to ameliorate mtDNA disease through multiple molecular mechanisms.
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Affiliation(s)
- Suraiya Haroon
- Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, PA 19104, USA
| | - Annie Li
- Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, PA 19104, USA
| | - Jaye L Weinert
- Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, PA 19104, USA
| | - Clark Fritsch
- Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, PA 19104, USA
| | - Nolan G Ericson
- Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | | | - Bart P Braeckman
- Laboratory of Aging Physiology and Molecular Evolution, Biology Department, Ghent University, 9000 Ghent, Belgium
| | - Cole M Haynes
- Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA
| | - Jason H Bielas
- Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Tali Gidalevitz
- Department of Biology, Drexel University, Philadelphia, PA 19104, USA
| | - Marc Vermulst
- Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, PA 19104, USA.
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41
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Wu WS, Huang WC, Brown JS, Zhang D, Song X, Chen H, Tu S, Weng Z, Lee HC. pirScan: a webserver to predict piRNA targeting sites and to avoid transgene silencing in C. elegans. Nucleic Acids Res 2019; 46:W43-W48. [PMID: 29897582 PMCID: PMC6030828 DOI: 10.1093/nar/gky277] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2018] [Accepted: 04/04/2018] [Indexed: 11/25/2022] Open
Abstract
pirScan is a web-based tool for identifying C. elegans piRNA-targeting sites within a given mRNA or spliced DNA sequence. The purpose of our tool is to allow C. elegans researchers to predict piRNA targeting sites and to avoid the persistent germline silencing of transgenes that has rendered many constructs unusable. pirScan fulfills this purpose by first enumerating the predicted piRNA-targeting sites present in an input sequence. This prediction can be exported in a tabular or graphical format. Subsequently, pirScan suggests silent mutations that can be introduced to the input sequence that would allow the modified transgene to avoid piRNA targeting. The user can customize the piRNA targeting stringency and the silent mutations that he/she wants to introduce into the sequence. The modified sequences can be re-submitted to be certain that any previously present piRNA-targeting sites are now absent and no new piRNA-targeting sites are accidentally generated. This revised sequence can finally be downloaded as a text file and/or visualized in a graphical format. pirScan is freely available for academic use at http://cosbi4.ee.ncku.edu.tw/pirScan/.
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Affiliation(s)
- Wei-Sheng Wu
- Department of Electrical Engineering, National Cheng Kung University, Tainan, Taiwan
| | - Wei-Che Huang
- Department of Electrical Engineering, National Cheng Kung University, Tainan, Taiwan
| | - Jordan S Brown
- Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA
| | - Donglei Zhang
- Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA.,Department of Biochemistry and Molecular Biology, Huazhong University of Science and Technology, Wuhan, Hubei, China
| | - Xiaoyan Song
- Department of Clinical Laboratory, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Hao Chen
- Program in Bioinformatics and Integrative Biology, University of Mass. Medical School, Worcester, MA 01605, USA
| | - Shikui Tu
- Program in Bioinformatics and Integrative Biology, University of Mass. Medical School, Worcester, MA 01605, USA.,Department of Computer Science and Engineering, Shanghai Jiao Tong University, Shanghai, China
| | - Zhiping Weng
- Program in Bioinformatics and Integrative Biology, University of Mass. Medical School, Worcester, MA 01605, USA
| | - Heng-Chi Lee
- Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA
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42
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Nance J, Frøkjær-Jensen C. The Caenorhabditis elegans Transgenic Toolbox. Genetics 2019; 212:959-990. [PMID: 31405997 PMCID: PMC6707460 DOI: 10.1534/genetics.119.301506] [Citation(s) in RCA: 112] [Impact Index Per Article: 18.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2019] [Accepted: 06/01/2019] [Indexed: 12/30/2022] Open
Abstract
The power of any genetic model organism is derived, in part, from the ease with which gene expression can be manipulated. The short generation time and invariant developmental lineage have made Caenorhabditis elegans very useful for understanding, e.g., developmental programs, basic cell biology, neurobiology, and aging. Over the last decade, the C. elegans transgenic toolbox has expanded considerably, with the addition of a variety of methods to control expression and modify genes with unprecedented resolution. Here, we provide a comprehensive overview of transgenic methods in C. elegans, with an emphasis on recent advances in transposon-mediated transgenesis, CRISPR/Cas9 gene editing, conditional gene and protein inactivation, and bipartite systems for temporal and spatial control of expression.
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Affiliation(s)
- Jeremy Nance
- Helen L. and Martin S. Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular Medicine, Department of Cell Biology, New York University School of Medicine, New York 10016
| | - Christian Frøkjær-Jensen
- King Abdullah University of Science and Technology (KAUST), Biological and Environmental Science and Engineering Division (BESE), KAUST Environmental Epigenetics Program (KEEP), Thuwal 23955-6900, Saudi Arabia
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43
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Lok JB. CRISPR/Cas9 Mutagenesis and Expression of Dominant Mutant Transgenes as Functional Genomic Approaches in Parasitic Nematodes. Front Genet 2019; 10:656. [PMID: 31379923 PMCID: PMC6646703 DOI: 10.3389/fgene.2019.00656] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2019] [Accepted: 06/21/2019] [Indexed: 12/13/2022] Open
Abstract
DNA transformation of parasitic nematodes enables novel approaches to validating predictions from genomic and transcriptomic studies of these important pathogens. Notably, proof of principle for CRISPR/Cas9 mutagenesis has been achieved in Strongyloides spp., allowing identification of molecules essential to the functions of sensory neurons that mediate behaviors comprising host finding, invasion, and location of predilection sites by parasitic nematodes. Likewise, CRISPR/Cas9 knockout of the developmental regulatory transcription factor Ss-daf-16 has validated its function in regulating morphogenesis of infective third-stage larvae in Strongyloides stercoralis. While encouraging, these studies underscore challenges that remain in achieving straightforward validation of essential intervention targets in parasitic nematodes. Chief among these is the likelihood that knockout of multifunctional regulators like Ss-DAF-16 or its downstream mediator, the nuclear receptor Ss-DAF-12, will produce phenotypes so complex as to defy interpretation and will render affected worms incapable of infecting their hosts, thus preventing establishment of stable mutant lines. Approaches to overcoming these impediments could involve refinements to current CRISPR/Cas9 methods in Strongyloides including regulatable Cas9 expression from integrated transgenes and CRISPR/Cas9 editing to ablate specific functional motifs in regulatory molecules without complete knockout. Another approach would express transgenes encoding regulatory molecules of interest with mutations designed to similarly ablate or degrade specific functional motifs such as the ligand binding domain of Ss-DAF-12 while preserving core functions such as DNA binding. Such mutant transgenes would be expected to exert a dominant interfering effect on their endogenous counterparts. Published reports validate the utility of such dominant-negative approaches in Strongyloides.
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Affiliation(s)
- James B Lok
- Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States
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Hu R, Li H, Lei Z, Han Q, Yu X, Zhou N, Zhang X, Mao Y, Wang X, Irwin DM, Niu G, Tan H. Construction of a sensitive pyrogen-testing cell model by site-specific knock-in of multiple genes. Biotechnol Bioeng 2019; 116:2652-2661. [PMID: 31180145 DOI: 10.1002/bit.27084] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2019] [Revised: 05/15/2019] [Accepted: 06/05/2019] [Indexed: 11/07/2022]
Abstract
A pyrogen test is crucial for evaluating the safety of drugs and medical equipment, especially those involved in injections. As existing pyrogen tests, including the rabbit pyrogen test, the limulus amoebocyte lysate (LAL) test and the monocyte activation test have limitations, development of new models for pyrogen testing is necessary. Here we develop a sensitive cell model for pyrogen test based on the lipopolysaccharides (LPS) signal pathway. TLR4, MD2, and CD14 play key roles in the LPS-mediated pyrogen reaction. We established a new TLR4/MD2/CD14-specific overexpressing knock-in cell model using the CRISPR/CAS9 technology and homologous recombination to detect LPS. Stimulation of our TLR4/CD14/MD2 knock-in cell line model with LPS leads to the release of the cytokines IL-6 and TNF-alpha, with a detection limit of 0.005 EU/ml, which is greatly lower than the lower limit of 0.015 EU/ml detected by the Tachypleus amebocyte lysate (TAL) assay.
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Affiliation(s)
- Ruobi Hu
- Department of Pharmacology, Health Science Center, Peking University, Beijing, China
| | - Hui Li
- Department of Pharmacology, Health Science Center, Peking University, Beijing, China
| | - Zhen Lei
- Beijing N&N Genetech Company, Ltd., Beijing, China
| | - Qing Han
- Department of Pharmacology, Health Science Center, Peking University, Beijing, China
| | - Xiuyan Yu
- Department of Pharmacology, Health Science Center, Peking University, Beijing, China
| | - Na Zhou
- Department of Pharmacology, Health Science Center, Peking University, Beijing, China
| | - Xuehui Zhang
- Department of Pharmacology, Health Science Center, Peking University, Beijing, China
| | - Yiqing Mao
- Department of Pharmacology, Health Science Center, Peking University, Beijing, China
| | - Xi Wang
- Department of Pharmacology, Health Science Center, Peking University, Beijing, China
| | - David M Irwin
- Department of Pharmacology, Health Science Center, Peking University, Beijing, China.,Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
| | - Gang Niu
- Beijing N&N Genetech Company, Ltd., Beijing, China
| | - Huanran Tan
- Department of Pharmacology, Health Science Center, Peking University, Beijing, China
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Vicencio J, Martínez-Fernández C, Serrat X, Cerón J. Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans. Genetics 2019; 211:1143-1154. [PMID: 30696716 PMCID: PMC6456308 DOI: 10.1534/genetics.119.301965] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2018] [Accepted: 01/25/2019] [Indexed: 12/27/2022] Open
Abstract
CRISPR-based genome-editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is more challenging. We have developed Nested CRISPR, a cloning-free ribonucleoprotein-driven method that robustly produces endogenous fluorescent reporters with EGFP, mCherry or wrmScarlet in Caenorhabditis elegans This method is based on the division of the fluorescent protein (FP) sequence in three fragments. In the first step, single-stranded DNA (ssDNA) donors (≤200 bp) are used to insert the 5' and 3' fragments of the FP in the locus of interest. In the second step, these sequences act as homology regions for homology-directed repair using a double-stranded DNA (dsDNA) donor (PCR product) containing the middle fragment, thus completing the FP sequence. In Nested CRISPR, the first step involving ssDNA donors is a well-established method that yields high editing efficiencies, and the second step is reliable because it uses universal CRISPR RNAs (crRNAs) and PCR products. We have also used Nested CRISPR in a nonessential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step. In the search for modifications to optimize the method, we tested synthetic single guide RNAs (sgRNAs), but did not observe a significant increase in efficiency. To streamline the approach, we combined all step 1 and step 2 reagents in a single injection and were successful in three of five loci tested with editing efficiencies of up to 20%. Finally, we discuss the prospects of this method in the future.
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Affiliation(s)
- Jeremy Vicencio
- Genes, Diseases, and Therapies Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, 08908 Barcelona, Spain
| | - Carmen Martínez-Fernández
- Genes, Diseases, and Therapies Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, 08908 Barcelona, Spain
| | - Xènia Serrat
- Genes, Diseases, and Therapies Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, 08908 Barcelona, Spain
| | - Julián Cerón
- Genes, Diseases, and Therapies Program, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, 08908 Barcelona, Spain
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Cheerambathur DK, Prevo B, Chow TL, Hattersley N, Wang S, Zhao Z, Kim T, Gerson-Gurwitz A, Oegema K, Green R, Desai A. The Kinetochore-Microtubule Coupling Machinery Is Repurposed in Sensory Nervous System Morphogenesis. Dev Cell 2019; 48:864-872.e7. [PMID: 30827898 PMCID: PMC6436928 DOI: 10.1016/j.devcel.2019.02.002] [Citation(s) in RCA: 34] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2018] [Revised: 11/05/2018] [Accepted: 01/31/2019] [Indexed: 12/19/2022]
Abstract
Dynamic coupling of microtubule ends to kinetochores, built on the centromeres of chromosomes, directs chromosome segregation during cell division. Here, we report that the evolutionarily ancient kinetochore-microtubule coupling machine, the KMN (Knl1/Mis12/Ndc80-complex) network, plays a critical role in neuronal morphogenesis. We show that the KMN network concentrates in microtubule-rich dendrites of developing sensory neurons that collectively extend in a multicellular morphogenetic event that occurs during C. elegans embryogenesis. Post-mitotic degradation of KMN components in sensory neurons disrupts dendritic extension, leading to patterning and functional defects in the sensory nervous system. Structure-guided mutations revealed that the molecular interface that couples kinetochores to spindle microtubules also functions in neuronal development. These results identify a cell-division-independent function for the chromosome-segregation machinery and define a microtubule-coupling-dependent event in sensory nervous system morphogenesis.
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Affiliation(s)
- Dhanya K Cheerambathur
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA 92093, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, San Diego, La Jolla, CA 92093, USA.
| | - Bram Prevo
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA 92093, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, San Diego, La Jolla, CA 92093, USA
| | - Tiffany-Lynn Chow
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA 92093, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, San Diego, La Jolla, CA 92093, USA
| | - Neil Hattersley
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA 92093, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, San Diego, La Jolla, CA 92093, USA
| | - Shaohe Wang
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA 92093, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, San Diego, La Jolla, CA 92093, USA
| | - Zhiling Zhao
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA 92093, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, San Diego, La Jolla, CA 92093, USA
| | - Taekyung Kim
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA 92093, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, San Diego, La Jolla, CA 92093, USA
| | - Adina Gerson-Gurwitz
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA 92093, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, San Diego, La Jolla, CA 92093, USA
| | - Karen Oegema
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA 92093, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, San Diego, La Jolla, CA 92093, USA
| | - Rebecca Green
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA 92093, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, San Diego, La Jolla, CA 92093, USA
| | - Arshad Desai
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA 92093, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, San Diego, La Jolla, CA 92093, USA.
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Mojumder S, Sawamura R, Murayama Y, Ogura T, Yamanaka K. Functional characterization of UBXN-6, a C-terminal cofactor of CDC-48, in C. elegans. Biochem Biophys Res Commun 2019; 509:462-468. [PMID: 30595383 DOI: 10.1016/j.bbrc.2018.12.155] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2018] [Accepted: 12/21/2018] [Indexed: 11/26/2022]
Abstract
CDC-48 is a AAA (ATPases associated with diverse cellular activities) chaperone and participates in a wide range of cellular activities. Its functional diversity is determined by differential binding of a variety of cofactors. In this study, we analyzed the physiological role of a CDC-48 cofactor UBXN-6 in Caenorhabditis elegans. The amount of UBXN-6 was markedly increased upon starvation, but not with the treatment of tunicamycin and rapamycin. The induction upon starvation is a unique characteristic for UBXN-6 among C-terminal cofactors of CDC-48. During starvation, lysosomal activity is triggered for rapid clearance of cellular materials. We observed the lysosomal activity by monitoring GLO-1::GFP, a marker for lysosome-related organelles. We found that more puncta of GLO-1::GFP were observed in the ubxn-6 deletion mutant after 12 h starvation compared with the wild-type strain. Taken together, we propose that UBXN-6 is involved in clearance of cellular materials upon starvation in C. elegans.
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Affiliation(s)
- Suman Mojumder
- Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto, 860-0811, Japan; Program for Leading Graduate Schools "HIGO Program", Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto, 860-8556, Japan
| | - Rie Sawamura
- Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto, 860-0811, Japan
| | - Yuki Murayama
- Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto, 860-0811, Japan
| | - Teru Ogura
- Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto, 860-0811, Japan; Program for Leading Graduate Schools "HIGO Program", Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto, 860-8556, Japan
| | - Kunitoshi Yamanaka
- Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto, 860-0811, Japan.
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Strategies for Efficient Genome Editing Using CRISPR-Cas9. Genetics 2019; 211:431-457. [PMID: 30504364 PMCID: PMC6366907 DOI: 10.1534/genetics.118.301775] [Citation(s) in RCA: 54] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2018] [Accepted: 11/29/2018] [Indexed: 02/07/2023] Open
Abstract
The targetable DNA endonuclease CRISPR-Cas9 has transformed analysis of biological processes by enabling robust genome editing in model and nonmodel organisms. Although rules directing Cas9 to its target DNA via a guide RNA are straightforward, wide variation occurs in editing efficiency and repair outcomes for both imprecise error-prone repair and precise templated repair. We found that imprecise and precise DNA repair from double-strand breaks (DSBs) is asymmetric, favoring repair in one direction. Using this knowledge, we designed RNA guides and repair templates that increased the frequency of imprecise insertions and deletions and greatly enhanced precise insertion of point mutations in Caenorhabditis elegans We also devised strategies to insert long (10 kb) exogenous sequences and incorporate multiple nucleotide substitutions at a considerable distance from DSBs. We expanded the repertoire of co-conversion markers appropriate for diverse nematode species. These selectable markers enable rapid identification of Cas9-edited animals also likely to carry edits in desired targets. Lastly, we explored the timing, location, frequency, sex dependence, and categories of DSB repair events by developing loci with allele-specific Cas9 targets that can be contributed during mating from either male or hermaphrodite germ cells. We found a striking difference in editing efficiency between maternally and paternally contributed genomes. Furthermore, imprecise repair and precise repair from exogenous repair templates occur with high frequency before and after fertilization. Our strategies enhance Cas9-targeting efficiency, lend insight into the timing and mechanisms of DSB repair, and establish guidelines for achieving predictable precise and imprecise repair outcomes with high frequency.
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ATM phosphorylation of the actin-binding protein drebrin controls oxidation stress-resistance in mammalian neurons and C. elegans. Nat Commun 2019; 10:486. [PMID: 30700723 PMCID: PMC6353951 DOI: 10.1038/s41467-019-08420-w] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2018] [Accepted: 12/27/2018] [Indexed: 12/13/2022] Open
Abstract
Drebrin (DBN) regulates cytoskeletal functions during neuronal development, and is thought to contribute to structural and functional synaptic changes associated with aging and Alzheimer’s disease. Here we show that DBN coordinates stress signalling with cytoskeletal dynamics, via a mechanism involving kinase ataxia-telangiectasia mutated (ATM). An excess of reactive oxygen species (ROS) stimulates ATM-dependent phosphorylation of DBN at serine-647, which enhances protein stability and accounts for improved stress resilience in dendritic spines. We generated a humanized DBN Caenorhabditis elegans model and show that a phospho-DBN mutant disrupts the protective ATM effect on lifespan under sustained oxidative stress. Our data indicate a master regulatory function of ATM-DBN in integrating cytosolic stress-induced signalling with the dynamics of actin remodelling to provide protection from synapse dysfunction and ROS-triggered reduced lifespan. They further suggest that DBN protein abundance governs actin filament stability to contribute to the consequences of oxidative stress in physiological and pathological conditions. Drebrin is an actin-binding protein known to play a role in neuronal dendritic spines but its precise regulation is unclear. Here, the authors report that DBN is activated by oxidative stress in an ATM-kinase dependent manner and increases resistance to oxidative stress in mice and in C. elegans.
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CRISPR/Cas9 Methodology for the Generation of Knockout Deletions in Caenorhabditis elegans. G3-GENES GENOMES GENETICS 2019; 9:135-144. [PMID: 30420468 PMCID: PMC6325907 DOI: 10.1534/g3.118.200778] [Citation(s) in RCA: 69] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
The Caenorhabditis elegans Gene Knockout Consortium is tasked with obtaining null mutations in each of the more than 20,000 open reading frames (ORFs) of this organism. To date, approximately 15,000 ORFs have associated putative null alleles. As there has been substantial success in using CRISPR/Cas9 in C. elegans, this appears to be the most promising technique to complete the task. To enhance the efficiency of using CRISPR/Cas9 to generate gene deletions in C. elegans we provide a web-based interface to access our database of guide RNAs (http://genome.sfu.ca/crispr). When coupled with previously developed selection vectors, optimization for homology arm length, and the use of purified Cas9 protein, we demonstrate a robust and effective protocol for generating deletions for this large-scale project. Debate and speculation in the larger scientific community concerning off-target effects due to non-specific Cas9 cutting has prompted us to investigate through whole genome sequencing the occurrence of single nucleotide variants and indels accompanying targeted deletions. We did not detect any off-site variants above the natural spontaneous mutation rate and therefore conclude that this modified protocol does not generate off-target events to any significant degree in C. elegans. We did, however, observe a number of non-specific alterations at the target site itself following the Cas9-induced double-strand break and offer a protocol for best practice quality control for such events.
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