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1
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Kavousinia P, Ahmadi MH, Sadeghian H, Hosseini Bafghi M. Therapeutic potential of CRISPR/CAS9 genome modification in T cell-based immunotherapy of cancer. Cytotherapy 2024; 26:436-443. [PMID: 38466263 DOI: 10.1016/j.jcyt.2024.02.014] [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: 11/26/2023] [Revised: 02/05/2024] [Accepted: 02/13/2024] [Indexed: 03/12/2024]
Abstract
Today, genome editing technologies like zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) are being used in clinical trials and the treatment of diseases like acquired immunodeficiency syndrome (AIDS) and cancer. CRISPR stands out as one of the most advanced tools for genome editing due to its simplicity and cost-effectiveness. It can selectively modify specific locations in the genome, offering new possibilities for treating human diseases. The CRISPR system uses ribonucleic acid-deoxyribonucleic acid (RNA-DNA) recognition to combat infections, regulate gene expression, and treat cancer. Chimeric antigen receptor (CAR) T-cell therapy, which uses T lymphocytes to eliminate cancer cells, can be improved by combining it with CRISPR technology. However, there are challenges in using CAR-T cells, including a lack of quantity and quality, exhaustion, neurotoxicity, cytokine release syndrome (CRS), B cell aplasia, tumor lysis syndrome, and anaphylaxis. Preclinical studies on CRISPR-edited CAR-T cells show promising results and targeting detrimental regulatory genes can enhance cancer treatment in the future.
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Affiliation(s)
- Pegah Kavousinia
- Department of Laboratory Sciences, Faculty of Paramedical and Rehabilitation Sciences, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Mohammad Hossein Ahmadi
- Department of Laboratory Sciences, Faculty of Paramedical and Rehabilitation Sciences, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Hamid Sadeghian
- Department of Laboratory Sciences, Faculty of Paramedical and Rehabilitation Sciences, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Mahdi Hosseini Bafghi
- Department of Laboratory Sciences, Faculty of Paramedical and Rehabilitation Sciences, Mashhad University of Medical Sciences, Mashhad, Iran.
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2
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Chen Z, Zheng S, Fu C. Shotgun knockdown of RNA by CRISPR-Cas13d in fission yeast. J Cell Sci 2023; 136:297260. [PMID: 36825467 DOI: 10.1242/jcs.260769] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2022] [Accepted: 02/15/2023] [Indexed: 02/25/2023] Open
Abstract
The CRISPR-Cas13d system has a single small effector protein that targets RNA and does not require the presence of a protospacer flanking site in the targeted transcript. These features make CRISPR-Cas13d an attractive system for RNA manipulation. Here, we report the successful implementation of the CRISPR-Cas13d system in fission yeast for RNA knockdown. A high effectiveness of the CRISPR-Cas13d system was ensured by using an array of CRISPR RNAs (crRNAs) that are flanked by two self-cleaving ribozymes and are expressed from an RNA polymerase II promoter. Given the repressible nature of the promoter, RNA knockdown by the CRISPR-Cas13d system is reversible. Moreover, using the CRISPR-Cas13d system, we identified an effective crRNA array targeting the transcript of gfp and the effectiveness was demonstrated by successful knockdown of the transcripts of noc4-gfp, bub1-gfp and ade6-gfp. In principle, the effective GFP crRNA array allows knockdown of any transcript carrying the GFP sequences. This new CRISPR-Cas13d-based toolkit is expected to have a wide range of applications in many aspects of biology, including dissection of gene function and visualization of RNA.
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Affiliation(s)
- Zhikai Chen
- MOE Key Laboratory for Cellular Dynamics & School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, China
| | - Shengnan Zheng
- MOE Key Laboratory for Cellular Dynamics & School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, China
| | - Chuanhai Fu
- MOE Key Laboratory for Cellular Dynamics & School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, China
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3
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Applying the CRISPR/Cas9 for treating human and animal diseases: a comprehensive review. ANNALS OF ANIMAL SCIENCE 2023. [DOI: 10.2478/aoas-2023-0009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/03/2023]
Abstract
Abstract
Recently, genome editing tools have been extensively used in many biomedical sciences. The gene editing system is applied to modify the DNA sequences in the cellular system to comprehend their physiological response. A developing genome editing technology like clustered regularly short palindromic repeats (CRISPR) is widely expended in medical sciences. CRISPR and CRISPR-associated protein 9 (CRISPR/Cas9) system is being exploited to edit any DNA mutations related to inherited ailments to investigate in animals (in vivo) and cell lines (in vitro). Remarkably, CRISPR/Cas9 could be employed to examine treatments of many human genetic diseases such as Cystic fibrosis, Tyrosinemia, Phenylketonuria, Muscular dystrophy, Parkinson’s disease, Retinoschisis, Hemophilia, β-Thalassemia and Atherosclerosis. Moreover, CRISPR/Cas9 was used for disease resistance such as Tuberculosis, Johne’s diseases, chronic enteritis, and Brucellosis in animals. Finally, this review discusses existing progress in treating hereditary diseases using CRISPR/Cas9 technology and the high points accompanying obstacles.
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4
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Basu U, Riaz Ahmed S, Bhat BA, Anwar Z, Ali A, Ijaz A, Gulzar A, Bibi A, Tyagi A, Nebapure SM, Goud CA, Ahanger SA, Ali S, Mushtaq M. A CRISPR way for accelerating cereal crop improvement: Progress and challenges. Front Genet 2023; 13:866976. [PMID: 36685816 PMCID: PMC9852743 DOI: 10.3389/fgene.2022.866976] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Accepted: 11/21/2022] [Indexed: 01/09/2023] Open
Abstract
Humans rely heavily on cereal grains as a key source of nutrients, hence regular improvement of cereal crops is essential for ensuring food security. The current food crisis at the global level is due to the rising population and harsh climatic conditions which prompts scientists to develop smart resilient cereal crops to attain food security. Cereal crop improvement in the past generally depended on imprecise methods like random mutagenesis and conventional genetic recombination which results in high off targeting risks. In this context, we have witnessed the application of targeted mutagenesis using versatile CRISPR-Cas systems for cereal crop improvement in sustainable agriculture. Accelerated crop improvement using molecular breeding methods based on CRISPR-Cas genome editing (GE) is an unprecedented tool for plant biotechnology and agriculture. The last decade has shown the fidelity, accuracy, low levels of off-target effects, and the high efficacy of CRISPR technology to induce targeted mutagenesis for the improvement of cereal crops such as wheat, rice, maize, barley, and millets. Since the genomic databases of these cereal crops are available, several modifications using GE technologies have been performed to attain desirable results. This review provides a brief overview of GE technologies and includes an elaborate account of the mechanisms and applications of CRISPR-Cas editing systems to induce targeted mutagenesis in cereal crops for improving the desired traits. Further, we describe recent developments in CRISPR-Cas-based targeted mutagenesis through base editing and prime editing to develop resilient cereal crop plants, possibly providing new dimensions in the field of cereal crop genome editing.
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Affiliation(s)
- Umer Basu
- Division of Entomology, ICAR-Indian Agricultural Research Institute, New Delhi, India
| | - Syed Riaz Ahmed
- Nuclear Institute for Agriculture and Biology College, Pakistan Institute of Engineering and Applied Sciences (NIAB-C, PIEAS), Faisalabad, Pakistan
| | | | - Zunaira Anwar
- Nuclear Institute for Agriculture and Biology College, Pakistan Institute of Engineering and Applied Sciences (NIAB-C, PIEAS), Faisalabad, Pakistan
| | - Ahmad Ali
- National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
| | - Aqsa Ijaz
- Nuclear Institute for Agriculture and Biology College, Pakistan Institute of Engineering and Applied Sciences (NIAB-C, PIEAS), Faisalabad, Pakistan
| | - Addafar Gulzar
- Division of Plant Pathology, Faculty of Agriculture, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Wadura Sopore, India
| | - Amir Bibi
- Department of Plant Breeding and Genetics, Faculty of Agriculture Sciences, University of Agriculture Faisalabad, Faisalabad, Pakistan
| | - Anshika Tyagi
- Department of Biotechnology, Yeungnam University, Gyeongsan, South Korea
| | - Suresh M. Nebapure
- Division of Entomology, ICAR-Indian Agricultural Research Institute, New Delhi, India
| | - Chengeshpur Anjali Goud
- Institute of Biotechnology, Professor Jayashanker Telangana State Agriculture University, Hyderabad, India
| | - Shafat Ahmad Ahanger
- Division of Plant Pathology, Faculty of Agriculture, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Wadura Sopore, India,*Correspondence: Shafat Ahmad Ahanger, ; Sajad Ali, ; Muntazir Mushtaq,
| | - Sajad Ali
- Department of Biotechnology, Yeungnam University, Gyeongsan, South Korea,*Correspondence: Shafat Ahmad Ahanger, ; Sajad Ali, ; Muntazir Mushtaq,
| | - Muntazir Mushtaq
- ICAR-National Bureau of Plant Genetic Resources, Division of Germplasm Evaluation, Pusa Campus, New Delhi, India,*Correspondence: Shafat Ahmad Ahanger, ; Sajad Ali, ; Muntazir Mushtaq,
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5
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Iwata T. Japan to Global Eye Genetics Consortium: Extending Research Collaboration for Inherited Eye Diseases. Asia Pac J Ophthalmol (Phila) 2022; 11:360-368. [PMID: 35904986 DOI: 10.1097/apo.0000000000000535] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2021] [Accepted: 03/24/2022] [Indexed: 12/24/2022] Open
Abstract
Japan Eye Genetics Consortium (JEGC) was established in 2011 to migrate research system to all-Japan structure for collecting phenotype-genotype information for inherited retinal diseases and other retinal diseases including hereditary optic neuropathy and hereditary glaucoma. Diagnostic team was assembled to maintain quality of diagnostic and to collect phenotype information to database in Tokyo Medical Center (TMC). Over the past 10 years, 1538 pedigree [2788 deoxyribonucleic acid (DNA) samples] was collected from 38 ophthalmology departments and eye hospitals. Whole exome analysis has improved diagnostic rate from ~17% in 2011 to 53% in 2021, with 27% of known variants, 18% of novel variants in known gene, 8% of potential novel disease-causing genes, and 47% of pedigree with unknown cause. Approximately 70% of Japanese patients were affected by novel mutation or by unknown cause. In 2014, Asian Eye Genetics Consortium (AEGC) was established by researchers from Hong Kong, India, Japan, and the US, later renamed to Global Eye Genetics Consortium (GEGC) to expand the idea of collaborative research on rare genetic eye diseases in Asia, Middle East, Africa, and South America. GEGC phenotype-genotype database, GenEye, was constructed to collect and catalog genetic eye diseases at global scale. Over 200 members from 30 countries, GEGC now has 200 members from 30 continents, performing scientific programs, young investigator visiting program, and GEGC organized session at the meetings of the Asia-Pacific Academy of Ophthalmology (APAO), The Association for Research in Vision and Ophthalmology (ARVO), All India Ophthalmological Society (AIOS), World Ophthalmology Congress (WOC), and International Society for Eye Research (ISER).
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Affiliation(s)
- Takeshi Iwata
- Molecular and Cellular Biology Division, National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Center, Tokyo, Japan
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6
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Xu Z, Wang Q, Zhong H, Jiang Y, Shi X, Yuan B, Yu N, Zhang S, Yuan X, Guo S, Yang Y. Carrier strategies boost the application of CRISPR/Cas system in gene therapy. EXPLORATION (BEIJING, CHINA) 2022; 2:20210081. [PMID: 37323878 PMCID: PMC10190933 DOI: 10.1002/exp.20210081] [Citation(s) in RCA: 54] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/04/2021] [Accepted: 01/06/2022] [Indexed: 02/05/2023]
Abstract
Emerging clustered regularly interspaced short palindromic repeat/associated protein (CRISPR/Cas) genome editing technology shows great potential in gene therapy. However, proteins and nucleic acids suffer from enzymatic degradation in the physiological environment and low permeability into cells. Exploiting carriers to protect the CRISPR system from degradation, enhance its targeting of specific tissues and cells, and reduce its immunogenicity is essential to stimulate its clinical applications. Here, the authors review the state-of-the-art CRISPR delivery systems and their applications, and describe strategies to improve the safety and efficacy of CRISPR mediated genome editing, categorized by three types of cargo formats, that is, Cas: single-guide RNA ribonucleoprotein, Cas mRNA and single-guide RNA, and Cas plasmid expressing CRISPR/Cas systems. The authors hope this review will help develop safe and efficient nanomaterial-based carriers for CRISPR tools.
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Affiliation(s)
- Zunkai Xu
- Key Laboratory of Functional Polymer Materials of Ministry of EducationState Key Laboratory of Medicinal Chemical Biology and Institute of Polymer ChemistryCollege of ChemistryNankai UniversityTianjinChina
| | - Qingnan Wang
- State Key Laboratory of Biotherapy and Cancer CenterWest China HospitalSichuan University and Collaborative Innovation CenterChengduChina
| | - Haiping Zhong
- Key Laboratory of Functional Polymer Materials of Ministry of EducationState Key Laboratory of Medicinal Chemical Biology and Institute of Polymer ChemistryCollege of ChemistryNankai UniversityTianjinChina
| | - Yaoyao Jiang
- Key Laboratory of Functional Polymer Materials of Ministry of EducationState Key Laboratory of Medicinal Chemical Biology and Institute of Polymer ChemistryCollege of ChemistryNankai UniversityTianjinChina
| | - Xiaoguang Shi
- Key Laboratory of Functional Polymer Materials of Ministry of EducationState Key Laboratory of Medicinal Chemical Biology and Institute of Polymer ChemistryCollege of ChemistryNankai UniversityTianjinChina
| | - Bo Yuan
- School of MedicineNankai UniversityTianjinChina
- Tianjin Key Laboratory of Ophthalmology and Visual ScienceTianjin Eye InstituteTianjin Eye HospitalTianjinChina
| | - Na Yu
- Translational Medicine CenterKey Laboratory of Molecular Target & Clinical PharmacologySchool of Pharmaceutical Sciences and The Second Affiliated HospitalGuangzhou Medical UniversityGuangzhouChina
| | - Shubiao Zhang
- Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of EducationDalian Minzu UniversityDalianChina
| | - Xiaoyong Yuan
- Tianjin Key Laboratory of Ophthalmology and Visual ScienceTianjin Eye InstituteTianjin Eye HospitalTianjinChina
- Clinical College of OphthalmologyTianjin Medical UniversityTianjinChina
| | - Shutao Guo
- Key Laboratory of Functional Polymer Materials of Ministry of EducationState Key Laboratory of Medicinal Chemical Biology and Institute of Polymer ChemistryCollege of ChemistryNankai UniversityTianjinChina
| | - Yang Yang
- State Key Laboratory of Biotherapy and Cancer CenterWest China HospitalSichuan University and Collaborative Innovation CenterChengduChina
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7
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Gene Editing for Improved Animal Welfare and Production Traits in Cattle: Will This Technology Be Embraced or Rejected by the Public? SUSTAINABILITY 2021. [DOI: 10.3390/su13094966] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Integrating technology into agricultural systems has gained considerable traction, particularly over the last half century. Agricultural systems that incorporate the public’s concerns regarding farm animal welfare are more likely to be socially accepted in the long term, a key but often forgotten component of sustainability. Gene editing is a tool that has received considerable attention in the last five years, given its potential capacity to improve farm animal health, welfare, and production efficiency. This study aimed to explore the attitudes of Brazilian citizens regarding the applications of gene editing in cattle that generate offspring without horns; are more resistant to heat; and have increased muscle tissue. Using a mixed-methods approach, we surveyed participants via face-to-face, using in-depth interviews (Study 1) and an online questionnaire containing closed-ended questions (Study 2). Overall, the acceptability of gene editing was low and in cases where support was given it was highly dependent on the type and purpose of the application proposed. Using gene editing to improve muscle tissue growth was viewed as less acceptable compared to using gene editing to reduce heat stress or to produce hornless cattle. Support declined when the application was perceived to harm animal welfare, to be profit motivated or to reinforce the status quo of intensive livestock systems. The acceptability of gene editing was reduced when perceptions of risks and benefits were viewed as unevenly or unfairly distributed among consumers, corporations, different types of farmers, and the animals. Interviewees did not consider gene editing a “natural” process, citing dissenting reasons such as the high degree of human interference and the acceleration of natural processes. Our findings raised several issues that may need to be addressed for gene editing to comply with the social pillar of sustainable agriculture.
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8
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TALEN outperforms Cas9 in editing heterochromatin target sites. Nat Commun 2021; 12:606. [PMID: 33504770 PMCID: PMC7840734 DOI: 10.1038/s41467-020-20672-5] [Citation(s) in RCA: 54] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Accepted: 11/19/2020] [Indexed: 01/01/2023] Open
Abstract
Genome editing critically relies on selective recognition of target sites. However, despite recent progress, the underlying search mechanism of genome-editing proteins is not fully understood in the context of cellular chromatin environments. Here, we use single-molecule imaging in live cells to directly study the behavior of CRISPR/Cas9 and TALEN. Our single-molecule imaging of genome-editing proteins reveals that Cas9 is less efficient in heterochromatin than TALEN because Cas9 becomes encumbered by local searches on non-specific sites in these regions. We find up to a fivefold increase in editing efficiency for TALEN compared to Cas9 in heterochromatin regions. Overall, our results show that Cas9 and TALEN use a combination of 3-D and local searches to identify target sites, and the nanoscopic granularity of local search determines the editing outcomes of the genome-editing proteins. Taken together, our results suggest that TALEN is a more efficient gene-editing tool than Cas9 for applications in heterochromatin.
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9
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Abstract
The CRISPR/Cas9 system has transformed how gene knockout and knock-in studies are performed in the lab, and it is poised to revolutionize medicine. However, one of the present limitations of this technology is its imperfect specificity. While CRISPR/Cas9 can be programmed to cut a specific DNA target sequence with relative precision, off-target sequence cleavage can occur in large genomes. Importantly, several techniques have recently been developed to measure CRISPR/Cas9 on- and off-target DNA cleavage in cells. Here, we present detailed protocols for evaluating the specificity of CRISPR/Cas9 and related systems in cells using both targeted-approaches, in which off-target sites are known a priori, and unbiased approaches which are able to identify off-target cleavage events throughout an entire genome. Together, these techniques can be used to assess the reliability of experimental models generated using CRISPR/Cas9 as well as the safety of therapeutics employing this technology.
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Affiliation(s)
| | - Juan Jovel
- The Applied Genomics Core, Office of Research, University of Alberta, Edmonton, AB, Canada
| | - Basil P Hubbard
- Department of Pharmacology, University of Alberta, Edmonton, AB, Canada.
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10
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Cromwell CR, Hubbard BP. In Vitro Assays for Comparing the Specificity of First- and Next-Generation CRISPR/Cas9 Systems. Methods Mol Biol 2021; 2162:215-232. [PMID: 32926385 DOI: 10.1007/978-1-0716-0687-2_12] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
CRISPR/Cas9 has revolutionized the ability to edit cellular DNA and is poised to transform the treatment of genetic diseases. One of the major concerns regarding its therapeutic use is the potential for off-target DNA cleavage, which could have detrimental consequences in vivo. To circumvent this, a number of strategies have been employed to develop next-generation CRISPR/Cas9 systems with improved specificity. These include the development of new protein variants of Cas9, as well as chemically modified guide RNA molecules. Here, we provide detailed protocols for two in vitro methods that enable the specificity of first- and next-generation CRISPR/Cas9 systems to be compared, and we demonstrate their applicability to evaluating chemically modified guide RNAs. One of these assays allows the specificity of different guide RNA/Cas9 complexes to be compared on a set of known off-target DNA sequences, while the second provides a broad specificity profile based on cleavage of a massive library of potential off-target DNA sequences. Collectively, these assays may be used to evaluate the specificity of different CRISPR/Cas9 systems on any DNA target sequence in a time- and cost-effective manner.
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Affiliation(s)
| | - Basil P Hubbard
- Department of Pharmacology, University of Alberta, Edmonton, AB, Canada.
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Pavlovic K, Tristán-Manzano M, Maldonado-Pérez N, Cortijo-Gutierrez M, Sánchez-Hernández S, Justicia-Lirio P, Carmona MD, Herrera C, Martin F, Benabdellah K. Using Gene Editing Approaches to Fine-Tune the Immune System. Front Immunol 2020; 11:570672. [PMID: 33117361 PMCID: PMC7553077 DOI: 10.3389/fimmu.2020.570672] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2020] [Accepted: 08/20/2020] [Indexed: 12/26/2022] Open
Abstract
Genome editing technologies not only provide unprecedented opportunities to study basic cellular system functionality but also improve the outcomes of several clinical applications. In this review, we analyze various gene editing techniques used to fine-tune immune systems from a basic research and clinical perspective. We discuss recent advances in the development of programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases. We also discuss the use of programmable nucleases and their derivative reagents such as base editing tools to engineer immune cells via gene disruption, insertion, and rewriting of T cells and other immune components, such natural killers (NKs) and hematopoietic stem and progenitor cells (HSPCs). In addition, with regard to chimeric antigen receptors (CARs), we describe how different gene editing tools enable healthy donor cells to be used in CAR T therapy instead of autologous cells without risking graft-versus-host disease or rejection, leading to reduced adoptive cell therapy costs and instant treatment availability for patients. We pay particular attention to the delivery of therapeutic transgenes, such as CARs, to endogenous loci which prevents collateral damage and increases therapeutic effectiveness. Finally, we review creative innovations, including immune system repurposing, that facilitate safe and efficient genome surgery within the framework of clinical cancer immunotherapies.
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Affiliation(s)
- Kristina Pavlovic
- Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada (Andalusian Regional Government), Health Sciences Technology Park, Granada, Spain
- Maimonides Institute of Biomedical Research in Cordoba (IMIBIC), Cellular Therapy Unit, Reina Sofia University Hospital, University of Cordoba, Cordoba, Spain
| | - María Tristán-Manzano
- Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada (Andalusian Regional Government), Health Sciences Technology Park, Granada, Spain
| | - Noelia Maldonado-Pérez
- Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada (Andalusian Regional Government), Health Sciences Technology Park, Granada, Spain
| | - Marina Cortijo-Gutierrez
- Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada (Andalusian Regional Government), Health Sciences Technology Park, Granada, Spain
| | - Sabina Sánchez-Hernández
- Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada (Andalusian Regional Government), Health Sciences Technology Park, Granada, Spain
| | - Pedro Justicia-Lirio
- Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada (Andalusian Regional Government), Health Sciences Technology Park, Granada, Spain
- LentiStem Biotech, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada (Andalusian Regional Government), Health Sciences Technology Park, Granada, Spain
| | - M. Dolores Carmona
- Maimonides Institute of Biomedical Research in Cordoba (IMIBIC), Cellular Therapy Unit, Reina Sofia University Hospital, University of Cordoba, Cordoba, Spain
| | - Concha Herrera
- Maimonides Institute of Biomedical Research in Cordoba (IMIBIC), Cellular Therapy Unit, Reina Sofia University Hospital, University of Cordoba, Cordoba, Spain
- Department of Hematology, Reina Sofía University Hospital, Córdoba, Spain
| | - Francisco Martin
- Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada (Andalusian Regional Government), Health Sciences Technology Park, Granada, Spain
| | - Karim Benabdellah
- Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada (Andalusian Regional Government), Health Sciences Technology Park, Granada, Spain
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12
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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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13
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Park DS, Kim SE, Koo DB, Kang MJ. Histone deacetylases inhibitor and RAD51 recombinase increase transcription activator-like effector nucleases-mediated homologous recombination on the bovine β-casein gene locus. ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES 2020; 33:1023-1033. [PMID: 32054213 PMCID: PMC7206376 DOI: 10.5713/ajas.19.0654] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/13/2019] [Accepted: 10/14/2019] [Indexed: 01/28/2023]
Abstract
OBJECTIVE The efficiency of the knock-in process is very important to successful gene editing in domestic animals. Recently, it was reported that transient loosening of the nucleosomal folding of transcriptionally inactive chromatin might have the potential to enhance homologous recombination efficiency. The objective of this study was to determine whether histone deacetylases (HDAC) inhibitor and RAD51 recombinase (RAD51) expression were associated with increased knock-in efficiency on the β-casein (bCSN2) gene locus in mammary alveolar-large T antigen (MAC-T) cells using the transcription activator-like effector nucleases (TALEN) system. METHODS MAC-T cells were treated with HDAC inhibitors, valproic acid, trichostatin A, or sodium butyrate for 24 h, then transfected with a knock-in vector, RAD51 expression vector and TALEN to target the bCSN2 gene. After 3 days of transfection, the knock-in efficiency was confirmed by polymerase chain reaction and DNA sequencing of the target site. RESULTS The level of HDAC 2 protein in MAC-T cells was decreased by treatment with HDAC inhibitors. The knock-in efficiency in MAC-T cells treated with HDAC inhibitors was higher than in cells not treated with inhibitors. However, the length of the homologous arm of the knock-in vector made no difference in the knock-in efficiency. Furthermore, DNA sequencing confirmed that the precision of the knock-in was more efficient in MAC-T cells treated with sodium butyrate. CONCLUSION These results indicate that chromatin modification by HDAC inhibition and RAD51 expression enhanced the homologous recombination efficiency on the bCSN2 gene locus in MAC-T cells.
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Affiliation(s)
- Da Som Park
- Department of Animal Science, Chonnam National University, Gwangju 61186, Korea
| | - Se Eun Kim
- Department of Animal Science, Chonnam National University, Gwangju 61186, Korea
| | - Deog-Bon Koo
- Department of Biotechnology, College of Engineering, Daegu University, Gyeongsan 38453, Korea
| | - Man-Jong Kang
- Department of Animal Science, Chonnam National University, Gwangju 61186, Korea
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14
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Hu NY, Chen YT, Wang Q, Jie W, Liu YS, You QL, Li ZL, Li XW, Reibel S, Pfrieger FW, Yang JM, Gao TM. Expression Patterns of Inducible Cre Recombinase Driven by Differential Astrocyte-Specific Promoters in Transgenic Mouse Lines. Neurosci Bull 2019; 36:530-544. [PMID: 31828740 DOI: 10.1007/s12264-019-00451-z] [Citation(s) in RCA: 47] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2019] [Accepted: 09/19/2019] [Indexed: 01/12/2023] Open
Abstract
Astrocytes are the most abundant cell type in the central nervous system (CNS). They provide trophic support for neurons, modulate synaptic transmission and plasticity, and contribute to neuronal dysfunction. Many transgenic mouse lines have been generated to obtain astrocyte-specific expression of inducible Cre recombinase for functional studies; however, the expression patterns of inducible Cre recombinase in these lines have not been systematically characterized. We generated a new astrocyte-specific Aldh1l1-CreERT2 knock-in mouse line and compared the expression pattern of Cre recombinase between this and five widely-used transgenic lines (hGfap-CreERT2 from The Jackson Laboratory and The Mutant Mouse Resource and Research Center, Glast-CreERT2, Cx30-CreERT2, and Fgfr3-iCreERT2) by crossing with Ai14 mice, which express tdTomato fluorescence following Cre-mediated recombination. In adult Aldh1l1-CreERT2:Ai14 transgenic mice, tdTomato was detected throughout the CNS, and five novel morphologically-defined types of astrocyte were described. Among the six evaluated lines, the specificity of Cre-mediated recombination was highest when driven by Aldh1l1 and lowest when driven by hGfap; in the latter mice, co-staining between tdTomato and NeuN was observed in the hippocampus and cortex. Notably, evident leakage was noted in Fgfr3-iCreERT2 mice, and the expression level of tdTomato was low in the thalamus when Cre recombinase expression was driven by Glast and in the capsular part of the central amygdaloid nucleus when driven by Cx30. Furthermore, tdTomato was clearly expressed in peripheral organs in four of the lines. Our results emphasize that the astrocyte-specific CreERT2 transgenic lines used in functional studies should be carefully selected.
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Affiliation(s)
- Neng-Yuan Hu
- State Key Laboratory of Organ Failure Research, Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangdong Key Laboratory of Psychiatric Disorders, Collaborative Innovation Center for Brain Science, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
| | - Ya-Ting Chen
- State Key Laboratory of Organ Failure Research, Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangdong Key Laboratory of Psychiatric Disorders, Collaborative Innovation Center for Brain Science, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
| | - Qian Wang
- State Key Laboratory of Organ Failure Research, Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangdong Key Laboratory of Psychiatric Disorders, Collaborative Innovation Center for Brain Science, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
| | - Wei Jie
- State Key Laboratory of Organ Failure Research, Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangdong Key Laboratory of Psychiatric Disorders, Collaborative Innovation Center for Brain Science, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
| | - Yi-Si Liu
- State Key Laboratory of Organ Failure Research, Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangdong Key Laboratory of Psychiatric Disorders, Collaborative Innovation Center for Brain Science, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
| | - Qiang-Long You
- State Key Laboratory of Organ Failure Research, Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangdong Key Laboratory of Psychiatric Disorders, Collaborative Innovation Center for Brain Science, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
| | - Ze-Lin Li
- State Key Laboratory of Organ Failure Research, Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangdong Key Laboratory of Psychiatric Disorders, Collaborative Innovation Center for Brain Science, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
| | - Xiao-Wen Li
- State Key Laboratory of Organ Failure Research, Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangdong Key Laboratory of Psychiatric Disorders, Collaborative Innovation Center for Brain Science, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
| | - Sophie Reibel
- Chronobiotron - UMS 3415, University of Strasbourg, 67084, Strasbourg, France
| | - Frank W Pfrieger
- Institute of Cellular and Integrative Neurosciences, CNRS UPR 3212, University of Strasbourg, 67084, Strasbourg, France
| | - Jian-Ming Yang
- State Key Laboratory of Organ Failure Research, Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangdong Key Laboratory of Psychiatric Disorders, Collaborative Innovation Center for Brain Science, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China.
| | - Tian-Ming Gao
- State Key Laboratory of Organ Failure Research, Key Laboratory of Mental Health of the Ministry of Education, Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangdong Key Laboratory of Psychiatric Disorders, Collaborative Innovation Center for Brain Science, Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China.
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15
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Trimidal SG, Benjamin R, Bae JE, Han MV, Kong E, Singer A, Williams TS, Yang B, Schiller MR. Can Designer Indels Be Tailored by Gene Editing?: Can Indels Be Customized? Bioessays 2019; 41:e1900126. [PMID: 31693213 PMCID: PMC7202862 DOI: 10.1002/bies.201900126] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2019] [Revised: 10/01/2019] [Indexed: 12/23/2022]
Abstract
Genome editing with engineered nucleases (GEENs) introduce site-specific DNA double-strand breaks (DSBs) and repairs DSBs via nonhomologous end-joining (NHEJ) pathways that eventually create indels (insertions/deletions) in a genome. Whether the features of indels resulting from gene editing could be customized is asked. A review of the literature reveals how gene editing technologies via NHEJ pathways impact gene editing. The survey consolidates a body of literature that suggests that the type (insertion, deletion, and complex) and the approximate length of indel edits can be somewhat customized with different GEENs and by manipulating the expression of key NHEJ genes. Structural data suggest that binding of GEENs to DNA may interfere with binding of key components of DNA repair complexes, favoring either classical- or alternative-NHEJ. The hypotheses have some limitations, but if validated, will enable scientists to better control indel makeup, holding promise for basic science and clinical applications of gene editing. Also see the video abstract here https://youtu.be/vTkJtUsLi3w.
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Affiliation(s)
- Sara G Trimidal
- School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
- Nevada Institute of Personalized Medicine, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
| | - Ronald Benjamin
- School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
- Nevada Institute of Personalized Medicine, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
| | - Ji Eun Bae
- School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
- Nevada Institute of Personalized Medicine, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
| | - Mira V Han
- School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
- Nevada Institute of Personalized Medicine, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
| | - Elizabeth Kong
- School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
- Nevada Institute of Personalized Medicine, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
| | - Aaron Singer
- School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
- Nevada Institute of Personalized Medicine, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
| | - Tyler S Williams
- School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
- Nevada Institute of Personalized Medicine, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
| | - Bing Yang
- Donald Danforth Plant Science Center, St. Louis, MO, 63132, USA
| | - Martin R Schiller
- School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
- Nevada Institute of Personalized Medicine, University of Nevada Las Vegas, Las Vegas, NV, 89154, USA
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16
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El-Kenawy A, Benarba B, Neves AF, de Araujo TG, Tan BL, Gouri A. Gene surgery: Potential applications for human diseases. EXCLI JOURNAL 2019; 18:908-930. [PMID: 31762718 PMCID: PMC6868916 DOI: 10.17179/excli2019-1833] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/20/2019] [Accepted: 10/09/2019] [Indexed: 12/13/2022]
Abstract
Gene therapy became in last decade a new emerging therapeutic era showing promising results against different diseases such as cancer, cardiovascular diseases, diabetes, and neurological disorders. Recently, the genome editing technique for eukaryotic cells called CRISPR-Cas (Clustered Regulatory Interspaced Short Palindromic Repeats) has enriched the field of gene surgery with enhanced applications. In the present review, we summarized the different applications of gene surgery for treating human diseases such as cancer, diabetes, nervous, and cardiovascular diseases, besides the molecular mechanisms involved in these important effects. Several studies support the important therapeutic applications of gene surgery in a large number of health disorders and diseases including β-thalassemia, cancer, immunodeficiencies, diabetes, and neurological disorders. In diabetes, gene surgery was shown to be effective in type 1 diabetes by triggering different signaling pathways. Furthermore, gene surgery, especially that using CRISPR-Cas possessed important application on diagnosis, screening and treatment of several cancers such as lung, liver, pancreatic and colorectal cancer. Nevertheless, gene surgery still presents some limitations such as the design difficulties and costs regarding ZFNs (Zinc Finger Nucleases) and TALENs (Transcription Activator-Like Effector Nucleases) use, off-target effects, low transfection efficiency, in vivo delivery-safety and ethical issues.
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Affiliation(s)
- Ayman El-Kenawy
- Department of Pathology, College of Medicine, Taif University, Saudi Arabia
- Department of Molecular Biology, GEBRI, University of Sadat City, P.O. Box 79, Sadat City, Egypt
| | - Bachir Benarba
- Laboratory Research on Biological Systems and Geomatics, Faculty of Nature and Life Sciences, University of Mascara, Algeria
| | - Adriana Freitas Neves
- Institute of Biotechnology, Molecular Biology Laboratory, Universidade Federal de Goias, Catalao, Brazil
| | - Thaise Gonçalves de Araujo
- Laboratory of Genetics and Biotechnology, Institute of Biotechnology, Federal University of Uberlandia, Patos de Minas, MG, Brazil
| | - Bee Ling Tan
- Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
| | - Adel Gouri
- Laboratory of Medical Biochemistry, Faculty of Medicine, University of Annaba, Algeria
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17
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Kojima A, Sakaue T, Okazaki M, Shikata F, Kurata M, Imai Y, Nakaoka H, Masumoto J, Uchita S, Izutani H. A simple mouse model of pericardial adhesions. J Cardiothorac Surg 2019; 14:124. [PMID: 31253183 PMCID: PMC6599257 DOI: 10.1186/s13019-019-0940-9] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2019] [Accepted: 06/17/2019] [Indexed: 11/15/2022] Open
Abstract
Background Postoperative pericardial adhesions are considered a risk factor for redo cardiac surgery. Several large- and medium-size animal models of pericardial adhesions have been reported, but small animal models for investigating the development of anti-adhesion materials and molecular mechanisms of this condition are lacking. In this study, we aimed to establish a simple mouse model of pericardial adhesions to address this gap. Methods We administered blood, minocycline, picibanil, and talc into the murine pericardial cavity via one-shot injection. Micro-computed tomography analyses of contrast agent-injected mice were carried out for methodological evaluation. We investigated various dosages and treatment durations for molecules identified to be inducers of pericardial adhesion. The adhesive grade was quantified by scoring the strength and volume of adhesion tissues at sacrificed time points. Histological staining with hematoxylin and eosin and Masson’s trichrome, and immunostaining for F4/80 or αSMA was performed to investigate the structural features of pericardial adhesions, and pathological features of the pericardial adhesion tissue were compared with human clinical specimens. Results Administration of talc resulted in the most extensive pericardial adhesions. Micro-computed tomography imaging data confirmed that accurate injection into the pericardial cavity was achieved. We found the optimal condition for the formation of strong pericardial adhesions to be injection of 2.5 mg/g talc for 2 weeks. Furthermore, histological analysis showed that talc administration led to an invasion of myofibroblasts and macrophages in the pericardial cavity and epicardium, consistent with pathological findings in patients with left ventricular assistive devices. Conclusions We successfully established a simple mouse model of talc-induced pericardial adhesions, which mimics human pathology and could contribute to solving the clinical issues related to pericardial adhesions.
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Affiliation(s)
- Ai Kojima
- Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime, 791-0295, Japan
| | - Tomohisa Sakaue
- Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime, 791-0295, Japan. .,Department of Cell Growth and Tumor Regulation, Proteo-Science Center (PROS), Shitsukawa, Toon, 791-0295, Ehime, Japan.
| | - Mikio Okazaki
- Department of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8558, Japan
| | - Fumiaki Shikata
- Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime, 791-0295, Japan.,Paediatric Cardiac Surgery, Queensland Children's Hospital, South Brisbane, QLD, Australia
| | - Mie Kurata
- Department of Pathology, Division of Analytical Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, 791-0295, Ehime, Japan.,Department of Pathology, Proteo-Science Center (PROS), Shitsukawa, Toon, 791-0295, Ehime, Japan
| | - Yuuki Imai
- Division of Integrative Pathophysiology Proteo-Science Center, Ehime University Graduate School of Medicine, Shitsukawa, Toon, 791-0295, Ehime, Japan
| | - Hirotomo Nakaoka
- Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime, 791-0295, Japan
| | - Junya Masumoto
- Department of Pathology, Division of Analytical Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, 791-0295, Ehime, Japan.,Department of Pathology, Proteo-Science Center (PROS), Shitsukawa, Toon, 791-0295, Ehime, Japan
| | - Shunji Uchita
- Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime, 791-0295, Japan
| | - Hironori Izutani
- Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime, 791-0295, Japan
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18
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Kurtz S, Petersen B. Pre-determination of sex in pigs by application of CRISPR/Cas system for genome editing. Theriogenology 2019; 137:67-74. [PMID: 31208775 DOI: 10.1016/j.theriogenology.2019.05.039] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
In livestock industries, one sex is usually preferred because of the impact on the production (e.g. milk from cows, eggs from laying hens). Furthermore, in pig production, the male-specific boar taint is a big hurdle for consumer acceptance. Consequently, a shift in the ratio towards the desired sex would be a great benefit. The most widely applied method for pre-determination of the sex is fluorescence-activated sperm sorting, which relies on the different DNA content of the X- and Y-chromosomal sperm. However, the successful practical adaption of this method depends on its ease of use. At present, sperm sexing via fluorescence-activated cell sorting (FACS) has only reached commercial application in cattle. Nevertheless, sperm sexing technology still needs to be improved with respect to efficiency and reliability, to obtain high numbers of sexed sperm and less invasive sperm treatment to avoid damage. New genome editing technologies such as Zinc finger nucleases (ZFN), Transcription-activator like endonucleases (TALENs) and the CRISPR/Cas system have emerged and offer great potential to affect determination of the sex at the genome level. The sex-determining region on the Y chromosome (SRY) serves as a main genetic switch of male gender development. It was previously shown that a knockout of the SRY gene in mice and rabbits displayed suppressed testis development in the fetal gonadal ridges resulting in a female phenotype. These new technologies hold great opportunities to pre-determine sex in pigs. However, further investigations are needed to exploit their full potential for practical application.
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Affiliation(s)
- Stefanie Kurtz
- Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Mariensee, Neustadt am Rübenberge, Germany
| | - Björn Petersen
- Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Mariensee, Neustadt am Rübenberge, Germany.
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19
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Phenotyping analysis of p53 knockout mice produced by gene editing and comparison with conventional p53 knockout mice. Genes Genomics 2019; 41:701-712. [PMID: 30989490 DOI: 10.1007/s13258-019-00785-y] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2018] [Accepted: 01/03/2019] [Indexed: 02/07/2023]
Abstract
BACKGROUND Knockout (KO) mice developed by homologous recombination (HR) have become useful tools to elucidate gene function. However, HR has low KO efficiency and is time-consuming, labor-intensive, and expensive. 'Gene editing' has received much attention for efficient genetic manipulation. OBJECTIVE As generation of KO mice is simplified, KO mice produced by HR can be feasibly reproduced using gene editing. However, phenotyping analysis and comparison between KO mice produced by these two techniques is necessary. METHODS We generated p53 KO mice through gene editing and compared their phenotype with the already reported HR-mediated p53 KO mice. RESULTS Tumors occurred in 36 (73%) of 49 homozygous KO mice and the mean age of occurrence was 23 weeks, with lymphoma (64%) and sarcoma (23%) being the most common. Tumors were also developed in 12 heterozygous mice and the mean age of occurrence was 40 weeks, with sarcoma (54%) and lymphoma (46%) in high proportion. Homozygotes had a mean life span of 157 ± 52 days and developmental abnormalities were found in females compared to in males (P < 0.05, P < 0.001). CONCLUSION We analyzed the basic phenotype of p53 KO mice and observed no significant difference from the conventional HR-mediated p53 KO mice.
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20
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Lau RW, Wang B, Ricardo SD. Gene editing of stem cells for kidney disease modelling and therapeutic intervention. Nephrology (Carlton) 2019; 23:981-990. [PMID: 29851168 DOI: 10.1111/nep.13410] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/26/2018] [Indexed: 12/13/2022]
Abstract
Recent developments in targeted gene editing have paved the way for the wide adoption of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nucleases (Cas9) as an RNA-guided molecular tool to modify the genome of eukaryotic cells of animals. Theoretically, the translation of CRISPR-Cas9 can be applied to the treatment of inherited or acquired kidney disease, kidney transplantation and genetic corrections of somatic cells from kidneys with inherited mutations, such as polycystic kidney disease. Human pluripotent stem cells have been used to generate an unlimited source of kidney progenitor cells or, when spontaneously differentiated into three-dimensional kidney organoids, to model kidney organogenesis or the pathogenesis of disease. Gene editing now allows for the tagging and selection of specific kidney cell types or disease-specific gene knock in/out, which enables more precise understanding of kidney organogenesis and genetic diseases. This review discusses the mechanisms of action, in addition to the advantages and disadvantages, of the three major gene editing technologies, namely, CRISPR-Cas9, zinc finger nucleases and transcription activator-like effector nucleases. The implications of using gene editing to better understand kidney disease is reviewed in detail. In addition, the ethical issues of gene editing, which could be easily neglected in the modern, fast-paced research environment, are highlighted.
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Affiliation(s)
- Ricky Wk Lau
- Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia
| | - Bo Wang
- Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia
| | - Sharon D Ricardo
- Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia
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21
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Audira G, Sarasamma S, Chen JR, Juniardi S, Sampurna BP, Liang ST, Lai YH, Lin GM, Hsieh MC, Hsiao CD. Zebrafish Mutants Carrying Leptin a (lepa) Gene Deficiency Display Obesity, Anxiety, Less Aggression and Fear, and Circadian Rhythm and Color Preference Dysregulation. Int J Mol Sci 2018; 19:ijms19124038. [PMID: 30551684 PMCID: PMC6320766 DOI: 10.3390/ijms19124038] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2018] [Revised: 12/05/2018] [Accepted: 12/11/2018] [Indexed: 01/14/2023] Open
Abstract
Leptin, a hormone secreted by peripheral adipose tissues, regulates the appetite in animals. Recently, evidence has shown that leptin also plays roles in behavioral response in addition to controlling appetite. In this study, we examined the potential function of leptin on non-appetite behaviors in zebrafish model. By using genome editing tool of Transcription activator-like effector nuclease (TALEN), we successfully knocked out leptin a (lepa) gene by deleting 4 bp within coding region to create a premature-translation stop. Morphological and appetite analysis showed the lepa KO fish display a phenotype with obese, good appetite and elevation of Agouti-related peptide (AgRP) and Ghrelin hormones, consistent with the canonical function of leptin in controlling food intake. By multiple behavior endpoint analyses, including novel tank, mirror biting, predator avoidance, social interaction, shoaling, circadian rhythm, and color preference assay, we found the lepa KO fish display an anxiogenic phenotype showing hyperactivity with rapid swimming, less freezing time, less fear to predator, loose shoaling area forming, and circadian rhythm and color preference dysregulations. Using biochemical assays, melatonin, norepinephrine, acetylcholine and serotonin levels in the brain were found to be significantly reduced in lepa KO fish, while the levels of dopamine, glycine and cortisol in the brain were significantly elevated. In addition, the brain ROS level was elevated, and the anti-oxidative enzyme catalase level was reduced. Taken together, by performing loss-of-function multiple behavior endpoint testing and biochemical analysis, we provide strong evidence for a critical role of lepa gene in modulating anxiety, aggression, fear, and circadian rhythm behaviors in zebrafish for the first time.
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Affiliation(s)
- Gilbert Audira
- Department of Chemistry, Chung Yuan Christian University, Chung-Li 32023, Taiwan.
- Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li 32023, Taiwan.
| | - Sreeja Sarasamma
- Department of Chemistry, Chung Yuan Christian University, Chung-Li 32023, Taiwan.
- Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li 32023, Taiwan.
| | - Jung-Ren Chen
- Department of Biological Science & Technology College of Medicine, I-Shou University, Kaohsiung, 82445, Taiwan.
| | - Stevhen Juniardi
- Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li 32023, Taiwan.
| | | | - Sung-Tzu Liang
- Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li 32023, Taiwan.
| | - Yu-Heng Lai
- Department of Chemistry, Chinese Culture University, Taipei 11114, Taiwan.
| | - Geng-Ming Lin
- Laboratory of Marine Biology and Ecology, Third Institute of Oceanography, State OceanicAdministration, Xiamen 361005, China.
| | - Ming-Chia Hsieh
- Division of Endocrinology and Metabolism, Department of Internal Medicine, Changhua Christian Hospital, Changhua 50094, Taiwan.
| | - Chung-Der Hsiao
- Department of Chemistry, Chung Yuan Christian University, Chung-Li 32023, Taiwan.
- Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li 32023, Taiwan.
- Center of Nanotechnology, Chung Yuan Christian University, Chung-Li 32023, Taiwan.
- Center of Biomedical Technology, Chung Yuan Christian University, Chung-Li 32023, Taiwan.
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22
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Tian H, Luo J, Zhang Z, Wu J, Zhang T, Busato S, Huang L, Song N, Bionaz M. CRISPR/Cas9-mediated Stearoyl-CoA Desaturase 1 (SCD1) Deficiency Affects Fatty Acid Metabolism in Goat Mammary Epithelial Cells. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2018; 66:10041-10052. [PMID: 30180552 DOI: 10.1021/acs.jafc.8b03545] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/08/2023]
Abstract
Stearoyl-CoA desaturase 1 (SCD1) is a fatty acid desaturase catalyzing cis-double-bond formation in the Δ9 position to produce monounsaturated fatty acids essential for the synthesis of milk fat. Previous studies using RNAi methods have provided support for a role of SCD1 in goat mammary epithelial cells (GMEC); however, RNAi presents several limitations that might preclude a truthful understanding of the biological function of SCD1. To explore the function of SCD1 on fatty acid metabolism in GMEC, we used CRISPR-Cas9-mediated SCD1 knockout through non-homologous end-joining (NHEJ) and homology-directed repair (HDR) pathways in GMEC. We successfully introduced nucleotide deletions and mutations in the SCD1 gene locus through the NHEJ pathway and disrupted its second exon via insertion of an EGFP-PuroR segment using the HDR pathway. In clones derived from the latter, gene- and protein-expression data indicated that we obtained a monoallelic SCD1 knockout. A T7EN1-mediated assay revealed no off-targets in the surveyed sites. The contents of triacylglycerol and cholesterol and the desaturase index were significantly decreased as a consequence of SCD1 knockout. The deletion of SCD1 decreased the expression of other genes involved in de novo fatty acid synthesis, including SREBF1 and FASN, as well the fatty acid transporters FABP3 and FABP4. The downregulation of these genes partly explains the decrease of intracellular triacylglycerols. Our results indicate a successful SCD1 knockout in goat mammary cells using CRISPR-Cas9. The demonstration of the successful use of CRISPR-Cas9 in GMEC is an important step to producing transgenic goats to study mammary biology in vivo.
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Affiliation(s)
- Huibin Tian
- Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology , Northwest A&F University , Yangling 712100 , China
| | - Jun Luo
- Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology , Northwest A&F University , Yangling 712100 , China
| | - Zhifei Zhang
- Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology , Northwest A&F University , Yangling 712100 , China
| | - Jiao Wu
- Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology , Northwest A&F University , Yangling 712100 , China
| | - Tianying Zhang
- Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology , Northwest A&F University , Yangling 712100 , China
| | - Sebastiano Busato
- Department of Animal and Rangeland Sciences , Oregon State University , Corvallis , Oregon 97331 , United States
| | - Lian Huang
- Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology , Northwest A&F University , Yangling 712100 , China
| | - Ning Song
- Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology , Northwest A&F University , Yangling 712100 , China
| | - Massimo Bionaz
- Department of Animal and Rangeland Sciences , Oregon State University , Corvallis , Oregon 97331 , United States
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Vasodilator-stimulated phosphoprotein (VASP) is not a major mediator of platelet aggregation, thrombogenesis, haemostasis, and antiplatelet effect of prasugrel in rats. Sci Rep 2018; 8:9955. [PMID: 29967338 PMCID: PMC6028634 DOI: 10.1038/s41598-018-28181-8] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2018] [Accepted: 06/18/2018] [Indexed: 01/29/2023] Open
Abstract
Vasodilator-stimulated phosphoprotein (VASP) is a member of actin regulatory proteins implicated in platelet adhesion. In addition, phosphorylation of VASP is utilised for the assessment of platelet reactivity in patients treated with P2Y12 receptor antagonists, a class of antiplatelet agents. However, the role of VASP in platelet aggregation, thrombogenesis, haemostasis, and the antiplatelet effect of P2Y12 receptor antagonists remains unclear. We investigated these effects using heterozygous and homozygous VASP knockout rats generated with a CRISPR/Cas9 system. Baseline characteristics, such as haematology and other biochemical parameters, were comparable among the genotypes. In vitro platelet aggregation stimulated by adenosine diphosphate (ADP) or collagen, P-selectin expression of rat platelets treated with ADP, and in vivo thrombocytopenia induced by collagen were also comparable among the genotypes. In addition, in vivo thrombogenesis in a ferric chloride-induced arterial thrombosis model and bleeding time were also comparable among the genotypes. Furthermore, the in vitro antiplatelet effect of prasugrel, a third-generation P2Y12 receptor antagonist, was unaffected by VASP knockout. Although phosphorylated VASP is still an important surrogate marker specific for P2Y12 antagonists, our findings demonstrate that VASP is not a major mediator of platelet aggregation, thrombogenesis, haemostasis, and the antiplatelet effect of prasugrel in rats.
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Wang D, Wang XW, Peng XC, Xiang Y, Song SB, Wang YY, Chen L, Xin VW, Lyu YN, Ji J, Ma ZW, Li CB, Xin HW. CRISPR/Cas9 genome editing technology significantly accelerated herpes simplex virus research. Cancer Gene Ther 2018; 25:93-105. [PMID: 29691470 DOI: 10.1038/s41417-018-0016-3] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2017] [Revised: 12/24/2017] [Accepted: 12/28/2017] [Indexed: 12/20/2022]
Abstract
Herpes simplex viruses (HSVs) are important pathogens and ideal for gene therapy due to its large genome size. Previous researches on HSVs were hampered because the technology to construct recombinant HSVs were based on DNA homology-dependent repair (HDR) and plaque assay, which are inefficient, laborious, and time-consuming. Fortunately, clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) recently provided the possibility to precisely, efficiently, and rapidly edit genomes and indeed is successfully being used in HSVs. Importantly, CRISPR/Cas9 technology increased HSV HDR efficiency exponentially by a 10,000-1,000,000 times when making recombinant HSVs, and its combination with flow cytometric technology made HSV recombination practically automatic. These may have a significant impact on virus and gene therapy researches. This review will summarize the latest development and molecular mechanisms of CRISPR/Cas9 genome editing technology and its recent application in HSVs.
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Affiliation(s)
- Dong Wang
- The Second Clinical Medical School, Yangtze University, 434023, Jingzhou, Hubei Province, China
- Center for Oncology, Yangtze University Health Science Center, 434023, Jingzhou, Hubei Province, China
| | - Xian-Wang Wang
- The Second Clinical Medical School, Yangtze University, 434023, Jingzhou, Hubei Province, China
- Center for Oncology, Yangtze University Health Science Center, 434023, Jingzhou, Hubei Province, China
| | - Xiao-Chun Peng
- The Second Clinical Medical School, Yangtze University, 434023, Jingzhou, Hubei Province, China
- Center for Oncology, Yangtze University Health Science Center, 434023, Jingzhou, Hubei Province, China
| | - Ying Xiang
- The Second Clinical Medical School, Yangtze University, 434023, Jingzhou, Hubei Province, China
- Center for Oncology, Yangtze University Health Science Center, 434023, Jingzhou, Hubei Province, China
| | - Shi-Bao Song
- The Second Clinical Medical School, Yangtze University, 434023, Jingzhou, Hubei Province, China
- Center for Oncology, Yangtze University Health Science Center, 434023, Jingzhou, Hubei Province, China
| | - Ying-Ying Wang
- The Second Clinical Medical School, Yangtze University, 434023, Jingzhou, Hubei Province, China
- Center for Oncology, Yangtze University Health Science Center, 434023, Jingzhou, Hubei Province, China
| | - Lin Chen
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Changsha Medical University, 410219, Changsha, Hunan Province, China
| | - Victoria W Xin
- Montgomery Blair High School, Silver Spring, MD, 20901-2451, USA
| | - Yan-Ning Lyu
- Institute for Infectious Diseases and Endemic Diseases Prevention and Control, Beijing Center for Diseases Prevention and Control, 100013, Beijing, China
| | - Jiafu Ji
- Department of Gastrointestinal Surgery, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 100142, Beijing, China
| | - Zhao-Wu Ma
- The Second Clinical Medical School, Yangtze University, 434023, Jingzhou, Hubei Province, China.
- Center for Oncology, Yangtze University Health Science Center, 434023, Jingzhou, Hubei Province, China.
| | - Cheng-Bin Li
- Department of Laboratory Medicine, Jingzhou Central Hospital, the Second Clinical Medical School, Yangtze University, 434023, Jingzhou, Hubei Province, China.
| | - Hong-Wu Xin
- The Second Clinical Medical School, Yangtze University, 434023, Jingzhou, Hubei Province, China.
- Center for Oncology, Yangtze University Health Science Center, 434023, Jingzhou, Hubei Province, China.
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Gene-knocked out chimeric antigen receptor (CAR) T cells: Tuning up for the next generation cancer immunotherapy. Cancer Lett 2018; 423:95-104. [DOI: 10.1016/j.canlet.2018.03.010] [Citation(s) in RCA: 47] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2018] [Revised: 03/06/2018] [Accepted: 03/07/2018] [Indexed: 12/15/2022]
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26
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Shen Y, Zhang J, Yu T, Qi C. Generation of PTEN knockout bone marrow mesenchymal stem cell lines by CRISPR/Cas9-mediated genome editing. Cytotechnology 2018; 70:783-791. [PMID: 29387984 DOI: 10.1007/s10616-017-0183-3] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2017] [Accepted: 12/15/2017] [Indexed: 12/15/2022] Open
Abstract
The tumor suppressor PTEN is involved in the regulation of cell proliferation, lineage determination, motility, adhesion and apoptosis. Loss of PTEN in the bone mesenchymal stem cells (BMSCs) was shown to change their function in the repair tissue. So far, the CRISPR/Cas9 system has been proven extremely simple and flexible. Using this system to manipulate PTEN gene editing could produce the PTEN-Knocking-out (PTEN-KO) strain. We knocked out PTEN in MSCs and validated the expression by PCR and Western blot. To clarify the changes in proliferation, CCK-8 assay was applied. In support, living cell proportion was assessed by Trypan blue staining. For osteogenic and adipogenic induction, cells were cultured in different media for 2 weeks. Oil red staining and alizarin red staining were performed for assessment of osteogenic or adipogenic differentiation. The expression of Id4, Runx2, ALP and PPARγ was examined by qPCR and immunocytochemistry staining. The PTEN-KO strain was identified by sequencing. The PTEN-KO cells had an increased cell viability and higher survival compared with the wild type. However, decreased expression of Runx2 and PPARγ was found in the PTEN loss strain after induction, and consistently decreased osteogenic or adipogenic differentiation was observed by alizarin and oil red staining. Together, PTEN-KO strain showed an increased proliferation capability but decreased multi-directional differentiation potential. When BMSCs serve as seed cells for tissue engineering, the PTEN gene may be used as an indicator.
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Affiliation(s)
- Youliang Shen
- Department of Orthopaedics, Jiao Zhou Central Hospital of Qingdao City, Qingdao, 266300, China
| | - Jingjing Zhang
- Department of Orthopaedics, Jiao Zhou Central Hospital of Qingdao City, Qingdao, 266300, China
| | - Tengbo Yu
- Orthopaedic Center, The Affiliated Hospital of Qingdao University, Qingdao, 266300, China
| | - Chao Qi
- Orthopaedic Center, The Affiliated Hospital of Qingdao University, Qingdao, 266300, China.
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27
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Elkon R, Agami R. Characterization of noncoding regulatory DNA in the human genome. Nat Biotechnol 2017; 35:732-746. [DOI: 10.1038/nbt.3863] [Citation(s) in RCA: 63] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2016] [Accepted: 03/31/2017] [Indexed: 12/22/2022]
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28
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Application of the gene editing tool, CRISPR-Cas9, for treating neurodegenerative diseases. Neurochem Int 2017; 112:187-196. [PMID: 28732771 DOI: 10.1016/j.neuint.2017.07.007] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2017] [Revised: 06/12/2017] [Accepted: 07/16/2017] [Indexed: 12/26/2022]
Abstract
Increased accumulation of transcribed protein from the damaged DNA and reduced DNA repair capability contributes to numerous neurological diseases for which effective treatments are lacking. Gene editing techniques provide new hope for replacing defective genes and DNA associated with neurological diseases. With advancements in using such editing tools as zinc finger nucleases (ZFNs), meganucleases, and transcription activator-like effector nucleases (TALENs), etc., scientists are able to design DNA-binding proteins, which can make precise double-strand breaks (DSBs) at the target DNA. Recent developments with the CRISPR-Cas9 gene-editing technology has proven to be more precise and efficient when compared to most other gene-editing techniques. Two methods, non-homologous end joining (NHEJ) and homology-direct repair (HDR), are used in CRISPR-Cas9 system to efficiently excise the defective genes and incorporate exogenous DNA at the target site. In this review article, we provide an overview of the CRISPR-Cas9 methodology, including its molecular mechanism, with a focus on how in this gene-editing tool can be used to counteract certain genetic defects associated with neurological diseases. Detailed understanding of this new tool could help researchers design specific gene editing strategies to repair genetic disorders in selective neurological diseases.
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Lehmann J, Seebode C, Smolorz S, Schubert S, Emmert S. XPF knockout via CRISPR/Cas9 reveals that ERCC1 is retained in the cytoplasm without its heterodimer partner XPF. Cell Mol Life Sci 2017; 74:2081-2094. [PMID: 28130555 PMCID: PMC11107539 DOI: 10.1007/s00018-017-2455-7] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2016] [Revised: 12/01/2016] [Accepted: 01/03/2017] [Indexed: 01/05/2023]
Abstract
The XPF/ERCC1 heterodimeric complex is essentially involved in nucleotide excision repair (NER), interstrand crosslink (ICL), and double-strand break repair. Defects in XPF lead to severe diseases like xeroderma pigmentosum (XP). Up until now, XP-F patient cells have been utilized for functional analyses. Due to the multiple roles of the XPF/ERCC1 complex, these patient cells retain at least one full-length allele and residual repair capabilities. Despite the essential function of the XPF/ERCC1 complex for the human organism, we successfully generated a viable immortalised human XPF knockout cell line with complete loss of XPF using the CRISPR/Cas9 technique in fetal lung fibroblasts (MRC5Vi cells). These cells showed a markedly increased sensitivity to UVC, cisplatin, and psoralen activated by UVA as well as reduced repair capabilities for NER and ICL repair as assessed by reporter gene assays. Using the newly generated knockout cells, we could show that human XPF is markedly involved in homologous recombination repair (HRR) but dispensable for non-homologous end-joining (NHEJ). Notably, ERCC1 was not detectable in the nucleus of the XPF knockout cells indicating the necessity of a functional XPF/ERCC1 heterodimer to allow ERCC1 to enter the nucleus. Overexpression of wild-type XPF could reverse this effect as well as the repair deficiencies.
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Affiliation(s)
- Janin Lehmann
- Clinic and Policlinic for Dermatology and Venereology, University Medical Centre Rostock, Strempelstrasse 13, 18057, Rostock, Germany
- Department of Dermatology, Venereology and Allergology, University Medical Centre Goettingen, Robert-Koch-Strasse 40, 37075, Goettingen, Germany
| | - Christina Seebode
- Clinic and Policlinic for Dermatology and Venereology, University Medical Centre Rostock, Strempelstrasse 13, 18057, Rostock, Germany
| | - Sabine Smolorz
- Department of Dermatology, Venereology and Allergology, University Medical Centre Goettingen, Robert-Koch-Strasse 40, 37075, Goettingen, Germany
| | - Steffen Schubert
- Department of Dermatology, Venereology and Allergology, University Medical Centre Goettingen, Robert-Koch-Strasse 40, 37075, Goettingen, Germany
| | - Steffen Emmert
- Clinic and Policlinic for Dermatology and Venereology, University Medical Centre Rostock, Strempelstrasse 13, 18057, Rostock, Germany.
- Department of Dermatology, Venereology and Allergology, University Medical Centre Goettingen, Robert-Koch-Strasse 40, 37075, Goettingen, Germany.
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30
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Zhang K, Raboanatahiry N, Zhu B, Li M. Progress in Genome Editing Technology and Its Application in Plants. FRONTIERS IN PLANT SCIENCE 2017; 8:177. [PMID: 28261237 PMCID: PMC5306361 DOI: 10.3389/fpls.2017.00177] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/16/2016] [Accepted: 01/27/2017] [Indexed: 05/19/2023]
Abstract
Genome editing technology (GET) is a versatile approach that has progressed rapidly as a mechanism to alter the genotype and phenotype of organisms. However, conventional genome modification using GET cannot satisfy current demand for high-efficiency and site-directed mutagenesis, retrofitting of artificial nucleases has developed into a new avenue within this field. Based on mechanisms to recognize target genes, newly-developed GETs can generally be subdivided into three cleavage systems, protein-dependent DNA cleavage systems (i.e., zinc-finger nucleases, ZFN, and transcription activator-like effector nucleases, TALEN), RNA-dependent DNA cleavage systems (i.e., clustered regularly interspaced short palindromic repeats-CRISPR associated proteins, CRISPR-Cas9, CRISPR-Cpf1, and CRISPR-C2c1), and RNA-dependent RNA cleavage systems (i.e., RNA interference, RNAi, and CRISPR-C2c2). All these techniques can lead to double-stranded (DSB) or single-stranded breaks (SSB), and result in either random mutations via non-homologous end-joining (NHEJ) or targeted mutation via homologous recombination (HR). Thus, site-directed mutagenesis can be induced via targeted gene knock-out, knock-in, or replacement to modify specific characteristics including morphology-modification, resistance-enhancement, and physiological mechanism-improvement along with plant growth and development. In this paper, an non-comprehensive review on the development of different GETs as applied to plants is presented.
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Affiliation(s)
- Kai Zhang
- Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and TechnologyWuhan, China
- Hubei Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains, Huanggang Normal UniversityHuanggang, China
| | - Nadia Raboanatahiry
- Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and TechnologyWuhan, China
| | - Bin Zhu
- Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and TechnologyWuhan, China
| | - Maoteng Li
- Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and TechnologyWuhan, China
- Hubei Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains, Huanggang Normal UniversityHuanggang, China
- *Correspondence: Maoteng Li
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Yucel D, Kocabas F. Developments in Hematopoietic Stem Cell Expansion and Gene Editing Technologies. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2017; 1079:103-125. [DOI: 10.1007/5584_2017_114] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
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32
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Abstract
Precise genome editing is a powerful tool for analysis of gene function. However, in spermatogonial stem cells (SSCs), this still remains a big challenge mainly due to low efficiency and complexity of currently available gene editing techniques. The CRISPR-Cas9 system from bacteria has been applied to modifying genome in different species at a very high efficiency and specificity. Here we describe CRISPR-Cas9-mediated gene editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in SSCs. This protocol provides guidelines for derivation of SSCs, nucleofection of SSCs with the CRISPR-Cas9 system, transplantation of the gene-modified SSCs into the recipient testes, and production of mice using transplanted SSC-derived round spermatids.
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Affiliation(s)
- Yinghua Wang
- State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 320 Yueyang Road, Shanghai, 200031, China
| | - Yifu Ding
- State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 320 Yueyang Road, Shanghai, 200031, China
| | - Jinsong Li
- State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 320 Yueyang Road, Shanghai, 200031, China.
- School of Life Science and Technology, ShanghaiTech University, 100 Haike Road, Shanghai, 201210, China.
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Impact of Gene Editing Tools, Like CRISPR/Cas9, on the Public Health Response to Disease Outbreaks. Disaster Med Public Health Prep 2016; 11:155-159. [PMID: 27640728 DOI: 10.1017/dmp.2016.123] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
The purpose of this communication is to explore the implications of genome editing techniques, such as CRISPR/Cas9, on public health-related responses to outbreaks of disease. The recent commercialization of genome editing techniques makes the creation and release of genetically altered pathogens a much easier task, increasing the possibility to the point of needing discussion. Three areas need to be addressed: predictions concerning potential genetic alterations, predictions and implications concerning the release of genetically altered pathogens, and the short- and long-term implications of the release of genetically altered pathogens. Full discourse on these topics among professionals in the area of public health will help to combat harm from the use of any genetically altered biologic weapons. The topics covered here include a review of the CRISPR/Cas9 gene editing technique, including a discussion of which possibilities utilize genome editing. We then address predictions about the application of gene alterations in the context of bioweapons. We discuss a few basic concepts about the evolution of an intentionally released genetically altered organism based on circumstances and patterns gleaned from observing nature in the hope that this will aid in the public health response to bioterrorism attack. (Disaster Med Public Health Preparedness. 2017;11:155-159).
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Yi L, Li J. CRISPR-Cas9 therapeutics in cancer: promising strategies and present challenges. Biochim Biophys Acta Rev Cancer 2016; 1866:197-207. [PMID: 27641687 DOI: 10.1016/j.bbcan.2016.09.002] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2016] [Revised: 09/13/2016] [Accepted: 09/14/2016] [Indexed: 01/05/2023]
Abstract
Cancer is characterized by multiple genetic and epigenetic alterations that drive malignant cell proliferation and confer chemoresistance. The ability to correct or ablate such mutations holds immense promise for combating cancer. Recently, because of its high efficiency and accuracy, the CRISPR-Cas9 genome editing technique has been widely used in cancer therapeutic explorations. Several studies used CRISPR-Cas9 to directly target cancer cell genomic DNA in cellular and animal cancer models which have shown therapeutic potential in expanding our anticancer protocols. Moreover, CRISPR-Cas9 can also be employed to fight oncogenic infections, explore anticancer drugs, and engineer immune cells and oncolytic viruses for cancer immunotherapeutic applications. Here, we summarize these preclinical CRISPR-Cas9-based therapeutic strategies against cancer, and discuss the challenges and improvements in translating therapeutic CRISPR-Cas9 into clinical use, which will facilitate better application of this technique in cancer research. Further, we propose potential directions of the CRISPR-Cas9 system in cancer therapy.
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Affiliation(s)
- Lang Yi
- National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, People's Republic of China; Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China; Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, People's Republic of China
| | - Jinming Li
- National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, People's Republic of China; Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China; Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, People's Republic of China.
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35
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Ren C, Liu X, Zhang Z, Wang Y, Duan W, Li S, Liang Z. CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.). Sci Rep 2016; 6:32289. [PMID: 27576893 PMCID: PMC5006071 DOI: 10.1038/srep32289] [Citation(s) in RCA: 145] [Impact Index Per Article: 16.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2016] [Accepted: 08/04/2016] [Indexed: 12/21/2022] Open
Abstract
The type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has been successfully applied to edit target genes in multiple plant species. However, it remains unknown whether this system can be used for genome editing in grape. In this study, we described genome editing and targeted gene mutation in ‘Chardonnay’ suspension cells and plants via the CRISPR/Cas9 system. Two single guide RNAs (sgRNAs) were designed to target distinct sites of the L-idonate dehydrogenase gene (IdnDH). CEL I endonuclease assay and sequencing results revealed the expected indel mutations at the target site, and a mutation frequency of 100% was observed in the transgenic cell mass (CM) as well as corresponding regenerated plants with expression of sgRNA1/Cas9. The majority of the detected mutations in transgenic CM were 1-bp insertions, followed by 1- to 3-nucleotide deletions. Off-target activities were also evaluated by sequencing the potential off-target sites, and no obvious off-target events were detected. Our results demonstrated that the CRISPR/Cas9 system is an efficient and specific tool for precise genome editing in grape.
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Affiliation(s)
- Chong Ren
- Beijing Key Laboratory of Grape Science and Enology and Key Laboratory of Plant Resource, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, PR China.,University of Chinese Academy of Sciences, Beijing 100049, PR China
| | - Xianju Liu
- Beijing Key Laboratory of Grape Science and Enology and Key Laboratory of Plant Resource, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, PR China.,University of Chinese Academy of Sciences, Beijing 100049, PR China
| | - Zhan Zhang
- Beijing Key Laboratory of Grape Science and Enology and Key Laboratory of Plant Resource, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, PR China.,University of Chinese Academy of Sciences, Beijing 100049, PR China
| | - Yi Wang
- Beijing Key Laboratory of Grape Science and Enology and Key Laboratory of Plant Resource, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, PR China.,University of Chinese Academy of Sciences, Beijing 100049, PR China
| | - Wei Duan
- Beijing Key Laboratory of Grape Science and Enology and Key Laboratory of Plant Resource, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, PR China
| | - Shaohua Li
- Beijing Key Laboratory of Grape Science and Enology and Key Laboratory of Plant Resource, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, PR China
| | - Zhenchang Liang
- Beijing Key Laboratory of Grape Science and Enology and Key Laboratory of Plant Resource, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, PR China.,Sino-Africa Joint Research Center, Chinese Academy of Sciences, Wuhan 430074, PR China
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Dreyer T, Nicholson S, Ely A, Arbuthnot P, Bloom K. Improved antiviral efficacy using TALEN-mediated homology directed recombination to introduce artificial primary miRNAs into DNA of hepatitis B virus. Biochem Biophys Res Commun 2016; 478:1563-8. [PMID: 27590580 DOI: 10.1016/j.bbrc.2016.08.152] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2016] [Accepted: 08/26/2016] [Indexed: 01/05/2023]
Abstract
Chronic infection with hepatitis B virus (HBV) remains an important global health problem. Currently licensed therapies have modest curative efficacy, which is as a result of their transient effects and limited action on the viral replication intermediate comprising covalently closed circular DNA (cccDNA). Gene editing with artificial HBV-specific endonucleases and use of artificial activators of the RNA interference pathway have shown anti-HBV therapeutic promise. Although results from these gene therapies are encouraging, maximizing durable antiviral effects is important. To address this goal, a strategy that entails combining gene editing with homology-directed DNA recombination (HDR), to introduce HBV-silencing artificial primary microRNAs (pri-miRs) into HBV DNA targets, is reported here. Previously described transcription activator-like effector nucleases (TALENs) that target the core and surface sequences of HBV were used to introduce double stranded breaks in the viral DNA. Simultaneous administration of donor sequences encoding artificial promoterless anti-HBV pri-miRs, with flanking arms that were homologous to sequences adjoining the TALENs' targets, augmented antiviral efficacy. Analysis showed targeted integration and the length of the flanking homologous arms of donor DNA had a minimal effect on antiviral efficiency. These results support the notion that gene editing and silencing may be combined to effect improved inhibition of HBV gene expression.
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Affiliation(s)
- Timothy Dreyer
- SA-MRC/Wits Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa
| | - Samantha Nicholson
- SA-MRC/Wits Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa
| | - Abdullah Ely
- SA-MRC/Wits Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa
| | - Patrick Arbuthnot
- SA-MRC/Wits Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa
| | - Kristie Bloom
- SA-MRC/Wits Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa.
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Yu B, Lu R, Yuan Y, Zhang T, Song S, Qi Z, Shao B, Zhu M, Mi F, Cheng Y. Efficient TALEN-mediated myostatin gene editing in goats. BMC DEVELOPMENTAL BIOLOGY 2016; 16:26. [PMID: 27461387 PMCID: PMC4962387 DOI: 10.1186/s12861-016-0126-9] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/08/2015] [Accepted: 07/15/2016] [Indexed: 12/27/2022]
Abstract
Background Myostatin (MSTN) encodes a negative regulator of skeletal muscle mass that might have applications for promoting muscle growth in livestock. In this study, we aimed to test whether targeted MSTN editing, mediated by transcription activator-like effector nucleases (TALENs), is a viable approach to create myostatin-modified goats (Capra hircus). Results We obtained a pair of TALENs (MTAL-2) that could recognize and cut the targeted MSTN site in the goat genome. Fibroblasts from pedigreed goats were co-transfected with MTAL-2, and 272 monoclonal cell strains were confirmed to have mono- or bi-allelic mutations in MSTN. Ten cell strains with different genotypes were used as donor cells for somatic cell nuclear transfer, which produced three cloned kids (K179/MSTN−/−, K52-2/MSTN+/−, and K52-1/MSTN+/+). Conclusions The results suggested that the MTAL-2 could disrupt MSTN efficiently in the goat genome. The mutated somatic cells could be used to produce MSTN-site mutated goats without developmental disruption. Thus, TALENs is an effective method for accurate genome editing to produce site-modified goats. Electronic supplementary material The online version of this article (doi:10.1186/s12861-016-0126-9) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Baoli Yu
- College of Veterinary Medicine, Yangzhou University, No. 12 Wenhui Road, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Rui Lu
- College of Veterinary Medicine, Yangzhou University, No. 12 Wenhui Road, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Yuguo Yuan
- College of Veterinary Medicine, Yangzhou University, No. 12 Wenhui Road, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Ting Zhang
- College of Veterinary Medicine, Yangzhou University, No. 12 Wenhui Road, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Shaozheng Song
- College of Veterinary Medicine, Yangzhou University, No. 12 Wenhui Road, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Zhengqiang Qi
- College of Veterinary Medicine, Yangzhou University, No. 12 Wenhui Road, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Bin Shao
- College of Veterinary Medicine, Yangzhou University, No. 12 Wenhui Road, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Mengmin Zhu
- College of Veterinary Medicine, Yangzhou University, No. 12 Wenhui Road, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Fei Mi
- College of Veterinary Medicine, Yangzhou University, No. 12 Wenhui Road, Yangzhou, 225009, Jiangsu Province, People's Republic of China
| | - Yong Cheng
- College of Veterinary Medicine, Yangzhou University, No. 12 Wenhui Road, Yangzhou, 225009, Jiangsu Province, People's Republic of China.
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Finnigan GC, Thorner J. mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence. G3 (BETHESDA, MD.) 2016; 6:2147-56. [PMID: 27185399 PMCID: PMC4938667 DOI: 10.1534/g3.116.029801] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/30/2016] [Accepted: 05/10/2016] [Indexed: 02/06/2023]
Abstract
Genome editing exploiting CRISPR/Cas9 has been adopted widely in academia and in the biotechnology industry to manipulate DNA sequences in diverse organisms. Molecular engineering of Cas9 itself and its guide RNA, and the strategies for using them, have increased efficiency, optimized specificity, reduced inappropriate off-target effects, and introduced modifications for performing other functions (transcriptional regulation, high-resolution imaging, protein recruitment, and high-throughput screening). Moreover, Cas9 has the ability to multiplex, i.e., to act at different genomic targets within the same nucleus. Currently, however, introducing concurrent changes at multiple loci involves: (i) identification of appropriate genomic sites, especially the availability of suitable PAM sequences; (ii) the design, construction, and expression of multiple sgRNA directed against those sites; (iii) potential difficulties in altering essential genes; and (iv) lingering concerns about "off-target" effects. We have devised a new approach that circumvents these drawbacks, as we demonstrate here using the yeast Saccharomyces cerevisiae First, any gene(s) of interest are flanked upstream and downstream with a single unique target sequence that does not normally exist in the genome. Thereafter, expression of one sgRNA and cotransformation with appropriate PCR fragments permits concomitant Cas9-mediated alteration of multiple genes (both essential and nonessential). The system we developed also allows for maintenance of the integrated, inducible Cas9-expression cassette or its simultaneous scarless excision. Our scheme-dubbed mCAL for " M: ultiplexing of C: as9 at A: rtificial L: oci"-can be applied to any organism in which the CRISPR/Cas9 methodology is currently being utilized. In principle, it can be applied to install synthetic sequences into the genome, to generate genomic libraries, and to program strains or cell lines so that they can be conveniently (and repeatedly) manipulated at multiple loci with extremely high efficiency.
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Affiliation(s)
- Gregory C Finnigan
- Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202
| | - Jeremy Thorner
- Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202
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Huang H, Wu Q. CRISPR Double Cutting through the Labyrinthine Architecture of 3D Genomes. J Genet Genomics 2016; 43:273-88. [PMID: 27210040 DOI: 10.1016/j.jgg.2016.03.006] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2016] [Revised: 03/03/2016] [Accepted: 03/16/2016] [Indexed: 02/06/2023]
Abstract
The genomes are organized into ordered and hierarchical topological structures in interphase nuclei. Within discrete territories of each chromosome, topologically associated domains (TADs) play important roles in various nuclear processes such as gene regulation. Inside TADs separated by relatively constitutive boundaries, distal elements regulate their gene targets through specific chromatin-looping contacts such as long-distance enhancer-promoter interactions. High-throughput sequencing studies have revealed millions of potential regulatory DNA elements, which are much more abundant than the mere ∼20,000 genes they control. The recently emerged CRISPR-Cas9 genome editing technologies have enabled efficient and precise genetic and epigenetic manipulations of genomes. The multiplexed and high-throughput CRISPR capabilities facilitate the discovery and dissection of gene regulatory elements. Here, we describe the applications of CRISPR for genome, epigenome, and 3D genome editing, focusing on CRISPR DNA-fragment editing with Cas9 and a pair of sgRNAs to investigate topological folding of chromatin TADs and developmental gene regulation.
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Affiliation(s)
- Haiyan Huang
- Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang, Shanghai 200240, China
| | - Qiang Wu
- Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang, Shanghai 200240, China.
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40
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Wen K, Bui T, Weiss M, Li G, Kocher J, Yang X, Jobst PM, Vaught T, Ramsoondar J, Ball S, Clark-Deener S, Ayares D, Yuan L. B-Cell-Deficient and CD8 T-Cell-Depleted Gnotobiotic Pigs for the Study of Human Rotavirus Vaccine-Induced Protective Immune Responses. Viral Immunol 2016; 29:112-27. [PMID: 26824402 PMCID: PMC4782039 DOI: 10.1089/vim.2015.0105] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Genetically modified pigs have become available recently. In this study, we established the gnotobiotic pig model of human rotavirus (HRV) infection using cloned pigs with homozygous disruption in the gene encoding immunoglobulin heavy chain (HCKO), which totally impairs B-cell development. To clarify importance of B cells and cytotoxic T cells in rotavirus immunity, CD8 cells in a subset of the pigs were depleted by injecting antipig CD8 antibodies and the immune phenotypes of all pigs were examined. HCKO pigs, CD8 cell-depleted HCKO pigs, and wild-type (WT) pigs were vaccinated with an attenuated HRV vaccine and challenged with virulent HRV. Protection against HRV infection and diarrhea was assessed postchallenge and detailed T-cell subset responses were determined pre- and postchallenge. Significantly longer duration of virus shedding was seen in vaccinated HCKO pigs than in WT pigs, indicating the importance of B cells in vaccine-induced protective immunity. Vaccinated HCKO/CD8(-) pigs shed significantly higher number of infectious virus than WT pigs and non-CD8-depleted HCKO pigs, indicating the importance of CD8 T cells in controlling virus replication. Therefore, both B cells and CD8 T cells play an important role in the protection against rotavirus infection. HCKO and HCKO/CD8(-) pigs did not differ significantly in diarrhea and virus shedding postchallenge; increased CD4 and CD8(-) γδ T-cell responses probably compensated partially for the lack of CD8 T cells. This study demonstrated that HCKO pigs can serve as a valuable model for dissection of protective immune responses against viral infections and diseases.
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Affiliation(s)
- Ke Wen
- Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
| | - Tammy Bui
- Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
| | - Mariah Weiss
- Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
| | - Guohua Li
- Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
| | - Jacob Kocher
- Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
| | - Xingdong Yang
- Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
| | - Peter M. Jobst
- Teaching & Research Animal Care Support Service, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
| | | | | | | | - Sherrie Clark-Deener
- Department of Large Animal Clinical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
| | | | - Lijuan Yuan
- Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
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Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is a prokaryotic adaptive immune system that is represented in most archaea and many bacteria. Among the currently known prokaryotic defense systems, the CRISPR-Cas genomic loci show unprecedented complexity and diversity. Classification of CRISPR-Cas variants that would capture their evolutionary relationships to the maximum possible extent is essential for comparative genomic and functional characterization of this theoretically and practically important system of adaptive immunity. To this end, a multipronged approach has been developed that combines phylogenetic analysis of the conserved Cas proteins with comparison of gene repertoires and arrangements in CRISPR-Cas loci. This approach led to the current classification of CRISPR-Cas systems into three distinct types and ten subtypes for each of which signature genes have been identified. Comparative genomic analysis of the CRISPR-Cas systems in new archaeal and bacterial genomes performed over the 3 years elapsed since the development of this classification makes it clear that new types and subtypes of CRISPR-Cas need to be introduced. Moreover, this classification system captures only part of the complexity of CRISPR-Cas organization and evolution, due to the intrinsic modularity and evolutionary mobility of these immunity systems, resulting in numerous recombinant variants. Moreover, most of the cas genes evolve rapidly, complicating the family assignment for many Cas proteins and the use of family profiles for the recognition of CRISPR-Cas subtype signatures. Further progress in the comparative analysis of CRISPR-Cas systems requires integration of the most sensitive sequence comparison tools, protein structure comparison, and refined approaches for comparison of gene neighborhoods.
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Abstract
Zebrafish is a valuable model organism to study vertebrate development, organ regeneration and to generate human disease models. As an important member of the arsenal of genome editing, TALE nucleases (TALENs) have implicated in broad applications in zebrafish reverse genetic studies. In this chapter, we describe the detailed protocols of TALEN-mediated genome manipulations in zebrafish, including targeted gene disruption by indel mutations, deletion of large genomic regions by using two pairs of TALENs, and precise genome modification by homologous recombination (HR).
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Affiliation(s)
- Peng Huang
- Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, College of Life Sciences, Peking University, Beijing, 100871, P. R. China
| | - An Xiao
- Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, College of Life Sciences, Peking University, Beijing, 100871, P. R. China
| | - Xiangjun Tong
- Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, College of Life Sciences, Peking University, Beijing, 100871, P. R. China
| | - Shuo Lin
- Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, CA, 90095, USA
| | - Bo Zhang
- Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, College of Life Sciences, Peking University, Beijing, 100871, P. R. China. .,College of Life Sciences, Peking University, No 5 YiHeYuan Rd., Haidian District, Beijing, 100871, P. R. China.
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Shi X, He BL, Ma ACH, Leung AYH. Fishing the targets of myeloid malignancies in the era of next generation sequencing. Blood Rev 2015; 30:119-30. [PMID: 26443083 DOI: 10.1016/j.blre.2015.09.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2015] [Revised: 08/15/2015] [Accepted: 09/04/2015] [Indexed: 11/29/2022]
Abstract
Recent advent in next generation sequencing (NGS) and bioinformatics has generated an unprecedented amount of genetic information in myeloidmalignancies. This information may shed lights to the pathogenesis, diagnosis and prognostication of these diseases and provide potential targets for therapeutic intervention. However, the rapid emergence of genetic information will quickly outpace their functional validation by conventional laboratory platforms. Foundational knowledge about zebrafish hematopoiesis accumulated over the past two decades and novel genomeediting technologies and research strategies in thismodel organismhavemade it a unique and timely research tool for the study of human blood diseases. Recent studies modeling human myeloid malignancies in zebrafish have also highlighted the technical feasibility and clinical relevance of thesemodels. Careful validation of experimental protocols and standardization among laboratorieswill further enhance the application of zebrafish in the scientific communities and provide important insights to the personalized treatment ofmyeloid malignancies.
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Affiliation(s)
- Xiangguo Shi
- Division of Haematology, Medical Oncology and Bone Marrow Transplantation, Department of Medicine, LKS Faculty Medicine, The University of Hong Kong.
| | - Bai-Liang He
- Division of Haematology, Medical Oncology and Bone Marrow Transplantation, Department of Medicine, LKS Faculty Medicine, The University of Hong Kong.
| | - Alvin C H Ma
- Division of Haematology, Medical Oncology and Bone Marrow Transplantation, Department of Medicine, LKS Faculty Medicine, The University of Hong Kong.
| | - Anskar Y H Leung
- Division of Haematology, Medical Oncology and Bone Marrow Transplantation, Department of Medicine, LKS Faculty Medicine, The University of Hong Kong.
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44
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Zhang Z, Lau SW, Zhang L, Ge W. Disruption of Zebrafish Follicle-Stimulating Hormone Receptor (fshr) But Not Luteinizing Hormone Receptor (lhcgr) Gene by TALEN Leads to Failed Follicle Activation in Females Followed by Sexual Reversal to Males. Endocrinology 2015; 156:3747-62. [PMID: 25993524 DOI: 10.1210/en.2015-1039] [Citation(s) in RCA: 120] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Gonadotropins are primary hormones that control vertebrate reproduction. In a recent study, we analyzed the impacts of FSH and LH on zebrafish reproduction by disrupting FSH and LH-β genes (fshb and lhb) using transcription activator-like effector nuclease (TALEN) technology. Using the same approach, we successfully deleted FSH and LH receptor genes (fshr and lhcgr) in the present study. In contrast to the deficiency of its cognate ligand FSH, the fshr-deficient females showed a complete failure of follicle activation with all ovarian follicles arrested at the primary growth-previtellogenic transition, which is the marker for puberty onset in females. Interestingly, after blockade at the primary growth stage for varying times, all females reversed to males, and all these males were fertile. In fshr-deficient males, spermatogenesis was normal in adults, but the initiation of spermatogenesis in juveniles was retarded. In contrast to fshr, the deletion of the lhcgr gene alone caused no obvious phenotypes in both males and females; however, double mutation of fshr and lhcgr resulted in infertile males. In summary, our results in the present study showed that Fshr was indispensable to folliculogenesis and the disruption of the fshr gene resulted in a complete failure of follicle activation followed by masculinization into males. In contrast, lhcgr does not seem to be essential to zebrafish reproduction in both males and females. Neither Fshr nor Lhcgr deficiency could phenocopy the deficiency of their cognate ligands FSH and LH, which is likely due to the fact that Fshr can be activated by both FSH and LH in the zebrafish.
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Affiliation(s)
- Zhiwei Zhang
- Centre of Reproduction, Development and Aging (Z.Z., W.G.), Faculty of Health Sciences, University of Macau, Taipa, Macau China; and School of Life Sciences (Z.Z., S.-W.L., L.Z., W.G.), The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
| | - Shuk-Wa Lau
- Centre of Reproduction, Development and Aging (Z.Z., W.G.), Faculty of Health Sciences, University of Macau, Taipa, Macau China; and School of Life Sciences (Z.Z., S.-W.L., L.Z., W.G.), The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
| | - Lingling Zhang
- Centre of Reproduction, Development and Aging (Z.Z., W.G.), Faculty of Health Sciences, University of Macau, Taipa, Macau China; and School of Life Sciences (Z.Z., S.-W.L., L.Z., W.G.), The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
| | - Wei Ge
- Centre of Reproduction, Development and Aging (Z.Z., W.G.), Faculty of Health Sciences, University of Macau, Taipa, Macau China; and School of Life Sciences (Z.Z., S.-W.L., L.Z., W.G.), The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
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Applications of TALENs and CRISPR/Cas9 in human cells and their potentials for gene therapy. Mol Biotechnol 2015; 56:681-8. [PMID: 24870618 DOI: 10.1007/s12033-014-9771-z] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
The newly developed TALENs and emerging CRISPR/Cas9 have spurred interests in the field of genome engineering because of their ease of customization and high-efficient site-specific cleavages. Although these novel technologies have been successfully used in many types of cells, it is of great importance to apply them in human-derived cells to further observe and evaluate their clinical potentials in gene therapy. Here, we review the working mechanism of TALEN and CRISPR/Cas9, their effectiveness and specificity in human cells, and current methods to enhance efficiency and reduce off-target effects. Besides, CCR5 gene was chosen as a target example to illustrate their clinical potentials. Finally, some questions are raised for future research and for researchers to consider when making a proper choice bases on different purposes.
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46
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Laufer BI, Singh SM. Strategies for precision modulation of gene expression by epigenome editing: an overview. Epigenetics Chromatin 2015; 8:34. [PMID: 26388942 PMCID: PMC4574080 DOI: 10.1186/s13072-015-0023-7] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2015] [Accepted: 08/19/2015] [Indexed: 01/27/2023] Open
Abstract
Genome editing technology has evolved rather quickly and become accessible to most researchers. It has resulted in far reaching implications and a number of novel designer systems including epigenome editing. Epigenome editing utilizes a combination of nuclease-null genome editing systems and effector domains to modulate gene expression. In particular, Zinc Finger, Transcription-Activator-Like Effector, and CRISPR/Cas9 have emerged as modular systems that can be modified to allow for precision manipulation of epigenetic marks without altering underlying DNA sequence. This review contains a comprehensive catalog of effector domains that can be used with components of genome editing systems to achieve epigenome editing. Ultimately, the evidence-based design of epigenome editing offers a novel improvement to the limited attenuation strategies. There is much potential for editing and/or correcting gene expression in somatic cells toward a new era of functional genomics and personalized medicine.
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Affiliation(s)
- Benjamin I Laufer
- Molecular Genetics Unit, Department of Biology, University of Western Ontario, London, ON Canada
| | - Shiva M Singh
- Molecular Genetics Unit, Department of Biology, University of Western Ontario, London, ON Canada
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47
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Yan Y, Wang H, Chen H, Lindström-Battle A, Jiao R. Ecdysone and Insulin Signaling Play Essential Roles in Readjusting the Altered Body Size Caused by the dGPAT4 Mutation in Drosophila. J Genet Genomics 2015; 42:487-94. [DOI: 10.1016/j.jgg.2015.06.008] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2015] [Revised: 06/04/2015] [Accepted: 06/04/2015] [Indexed: 12/19/2022]
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Wen D, Danquah M, Chaudhary AK, Mahato RI. Small molecules targeting microRNA for cancer therapy: Promises and obstacles. J Control Release 2015; 219:237-247. [PMID: 26256260 DOI: 10.1016/j.jconrel.2015.08.011] [Citation(s) in RCA: 79] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2015] [Revised: 07/20/2015] [Accepted: 08/04/2015] [Indexed: 02/06/2023]
Abstract
Aberrant expression of miRNAs is critically implicated in cancer initiation and progression. Therapeutic approaches focused on regulating miRNAs are therefore a promising approach for treating cancer. Antisense oligonucleotides, miRNA sponges, and CRISPR/Cas9 genome editing systems are being investigated as tools for regulating miRNAs. Despite the accruing insights in the use of these tools, delivery concerns have mitigated clinical application of such systems. In contrast, little attention has been given to the potential of small molecules to modulate miRNA expression for cancer therapy. In these years, many researches proved that small molecules targeting cancer-related miRNAs might have greater potential for cancer treatment. Small molecules targeting cancer related miRNAs showed significantly promising results in different cancer models. However, there are still several obstacles hindering the progress and clinical application in this area. This review discusses the development, mechanisms and application of small molecules for modulating oncogenic miRNAs (oncomiRs). Attention has also been given to screening technologies and perspectives aimed to facilitate clinical translation for small molecule-based miRNA therapeutics.
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Affiliation(s)
- Di Wen
- Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA
| | - Michael Danquah
- Department of Pharmaceutical Sciences, Chicago State University, 9501 South King Drive., Chicago, IL 60628, USA
| | - Amit Kumar Chaudhary
- Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA
| | - Ram I Mahato
- Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA.
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Tu Z, Yang W, Yan S, Guo X, Li XJ. CRISPR/Cas9: a powerful genetic engineering tool for establishing large animal models of neurodegenerative diseases. Mol Neurodegener 2015; 10:35. [PMID: 26238861 PMCID: PMC4524001 DOI: 10.1186/s13024-015-0031-x] [Citation(s) in RCA: 88] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2015] [Accepted: 07/24/2015] [Indexed: 02/07/2023] Open
Abstract
Animal models are extremely valuable to help us understand the pathogenesis of neurodegenerative disorders and to find treatments for them. Since large animals are more like humans than rodents, they make good models to identify the important pathological events that may be seen in humans but not in small animals; large animals are also very important for validating effective treatments or confirming therapeutic targets. Due to the lack of embryonic stem cell lines from large animals, it has been difficult to use traditional gene targeting technology to establish large animal models of neurodegenerative diseases. Recently, CRISPR/Cas9 was used successfully to genetically modify genomes in various species. Here we discuss the use of CRISPR/Cas9 technology to establish large animal models that can more faithfully mimic human neurodegenerative diseases.
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Affiliation(s)
- Zhuchi Tu
- Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
| | - Weili Yang
- Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
| | - Sen Yan
- Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
| | - Xiangyu Guo
- Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China.
| | - Xiao-Jiang Li
- Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China.
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, 30322, USA.
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50
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Li J, Shou J, Guo Y, Tang Y, Wu Y, Jia Z, Zhai Y, Chen Z, Xu Q, Wu Q. Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9. J Mol Cell Biol 2015; 7:284-98. [PMID: 25757625 PMCID: PMC4524425 DOI: 10.1093/jmcb/mjv016] [Citation(s) in RCA: 99] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2015] [Accepted: 03/02/2015] [Indexed: 12/26/2022] Open
Abstract
The human genome contains millions of DNA regulatory elements and a large number of gene clusters, most of which have not been tested experimentally. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) programed with a synthetic single-guide RNA (sgRNA) emerges as a method for genome editing in virtually any organisms. Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs. Specifically, we found that, in cultured human cells and mice, efficient precise inversions of DNA fragments ranging in size from a few tens of bp to hundreds of kb could be generated. In addition, DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks (DSBs) on two homologous chromosomes (chromatids). Moreover, junctions of combinatorial inversions and duplications of the protocadherin (Pcdh) gene clusters induced by Cas9 with four sgRNAs could be detected. In mice, we obtained founders with alleles of precise inversions, duplications, and deletions of DNA fragments of variable sizes by CRISPR. Interestingly, we found that very efficient inversions were mediated by microhomology-mediated end joining (MMEJ) through short inverted repeats. We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice. Finally, we applied this CRISPR method to a regulatory element of the Pcdhα cluster and found a new role in the regulation of members of the Pcdhγ cluster. This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters.
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Affiliation(s)
- Jinhuan Li
- Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Bio-X Center, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China
| | - Jia Shou
- Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Bio-X Center, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China
| | - Ya Guo
- Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Bio-X Center, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China
| | - Yuanxiao Tang
- Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Bio-X Center, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China
| | - Yonghu Wu
- Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Bio-X Center, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China
| | - Zhilian Jia
- Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Bio-X Center, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China
| | - Yanan Zhai
- Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Bio-X Center, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China
| | - Zhifeng Chen
- Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Bio-X Center, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China
| | - Quan Xu
- Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Bio-X Center, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China
| | - Qiang Wu
- Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Bio-X Center, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China
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