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Sampaio Cruz M, Manso AM, Soto-Hermida A, Bushway P, Silver E, Gunes BB, Tang Z, Gonzalez G, Lau S, Arbayo J, Najor RH, Chi L, Gu Y, Feng W, Cowling RT, Gustafsson AB, Chen J, Adler ED. Overlapping functions between Lamp2a and Lamp2b in cardiac autophagy. Autophagy 2025:1-12. [PMID: 40202173 DOI: 10.1080/15548627.2025.2484620] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2023] [Revised: 03/17/2025] [Accepted: 03/22/2025] [Indexed: 04/10/2025] Open
Abstract
LAMP2 is a ubiquitously expressed protein critical for autophagy. Alternative splicing gives rise to three isoforms. However, the roles of major LAMP2 isoforms in the heart are not known. To address this knowledge gap, we generated lamp2a and lamp2b knockout (KO) mice to investigate the role of these isoforms in heart function and autophagy. Deletion of either Lamp2a or Lamp2b did not alter cardiac structure or function. Lack of all LAMP2 isoforms led to increased cardiac fibrosis and reduced survival during pressure overload, which were not observed in lamp2a or lamp2b KO mice. Also, LAMP2B loss did not affect levels of the autophagy markers LC3-II and SQSTM1/p62. Conversely, LAMP2A was upregulated in hearts lacking LAMP2B, potentially preserving autophagy and cardiac function. Reintroducing LAMP2A in lamp2 KO mice effectively reduced autophagosome accumulation and improved cardiac function. Overall, these data support LAMP2 isoform functional redundancy in the myocardium under pathological conditions.Abbreviations: AAV: adeno-associated virus; ACTA2: actin alpha 2, smooth muscle, aorta; CMA: chaperone-mediated autophagy; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; LV: Left ventricle; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NPPA: natriuretic peptide type A; NPPB: natriuretic peptide type B; SQSTM1/p62: sequestosome 1; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; TAC: transverse aortic constriction; WT: wild type.
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Affiliation(s)
- Marina Sampaio Cruz
- Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA
| | - Ana Maria Manso
- Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA
| | - Angel Soto-Hermida
- Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA
| | - Paul Bushway
- Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA
| | - Elizabeth Silver
- Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA
| | - Betul Beyza Gunes
- Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA
| | - Zhiyuan Tang
- Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA
| | - Giovanni Gonzalez
- Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA
| | - Sharon Lau
- Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA
| | - Jordan Arbayo
- Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA
| | - Rita H Najor
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA
- Department of Pharmacology, University of California San Diego, La Jolla, CA, USA
| | - Liguo Chi
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA
- Department of Pharmacology, University of California San Diego, La Jolla, CA, USA
| | - Yusu Gu
- Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA
| | - Wei Feng
- Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA
| | - Randy T Cowling
- Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA
| | - Asa B Gustafsson
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA
- Department of Pharmacology, University of California San Diego, La Jolla, CA, USA
| | - Ju Chen
- Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA
| | - Eric D Adler
- Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, CA, USA
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Wu T, Chen Z, Zhang Z, Zhou X, Gu Y, Dinenno FA, Chen J. RBPMS and RBPMS2 Cooperate to Safeguard Cardiac Splicing. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.11.07.622565. [PMID: 39574760 PMCID: PMC11581027 DOI: 10.1101/2024.11.07.622565] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2025]
Abstract
Background Mutations in cardiac splicing factors (SFs) cause cardiomyopathy and congenital heart disease, underscoring the critical role of SFs in cardiac development and disease. Cardiac SFs are implicated to cooperatively regulate the splicing of essential cardiac genes, but the functional importance of their collaboration remains unclear. RNA Binding Protein with Multiple Splicing (RBPMS) and RBPMS2 are SFs involved in heart development and exhibit similar splicing regulatory activities in vitro , but it is unknown whether they cooperate to regulate splicing in vivo . Methods Rbpms and Rbpms2 single or double cardiomyocyte (CM)-specific knockout (KO) mice were generated and analyzed for cardiac phenotypes. RNA sequencing was performed to assess gene expression and splicing changes in single and double KOs. In silico analyses were used to dissect the mechanisms underlying distinct and overlapping roles of RBPMS and RBPMS2 in heart development. Results Mice lacking both RBPMS and RBPMS2 in CMs died before embryonic day 13.5 and developed sarcomere disarray, whereas Rbpms or Rbpms2 single CM-specific KO mice had normal sarcomere assembly and survived to adulthood. Defective sarcomere assembly is likely owing to the widespread mis-splicing of genes essential for cardiac contraction in double KO mice, underscoring the overlapping role of RBPMS and RBPMS2 in splicing regulation. Mechanistically, we found RBPMS and RBPMS collectively promote cardiac splicing program while repressing non-cardiac splicing programs. Moreover, RNA splicing maps suggested that the binding location of RBPMS and RBPMS2 on pre-mRNA dictates whether they function as splicing activators or repressors. Lastly, the requirement for RBPMS and/or RBPMS2 for splicing regulation arises from intrinsic features of the target exons. Conclusions Our results demonstrate that RBPMS and RBPMS2 work in concert to safeguard the splicing of genes essential for cardiac contraction, highlighting the importance of SF collaboration in maintaining cardiac splicing signature, which should be taken into consideration when devising future therapeutic approaches through modulating the activity of SFs. Novelty and Significance What Is Known?: Mutations in cardiac splicing factors (SFs) cause cardiomyopathy and congenital heart disease, and the splicing of cardiac genes is regulated by multiple SFs. However, the functional importance of the collaboration among specific cardiac SFs is unknown.RBPMS has emerged as a cardiac SF for sarcomere genes but is not required for sarcomere assembly. RBPMS2 can substitute RBPMS in in vitro splicing assays, yet its role in mammalian cardiomyocytes (CMs) remains unclear. What New Information Does This Article Contribute?: RBPMS and RBPMS2 have both distinct and overlapping roles in CMs.RBPMS and RBPMS2 collectively contribute to the maintenance of cardiac splicing program, which is essential for sarcomere assembly and embryonic survival.RNA splicing map of RBPMS and RBPMS2 reveals that they can function either as splicing activators or repressors, depending on their binding locations on pre-mRNA. This study provides compelling evidence of cooperation between cardiac splicing factors during heart development, which, to our knowledge, has not been demonstrated in vivo . Rbpms and Rbpms2 CM-specific double KO mice die in utero and exhibit sarcomere disarray, whereas single KO mice survive to adulthood with normal sarcomere structure but manifest distinct cardiac phenotypes, suggesting RBPMS and RBPMS2 possess both distinct and overlapping functions in CMs. Although mis-splicing in cardiac genes can be seen in all three KOs, the splicing signature of double KO hearts drastically shifts towards non-cardiac tissues, including more prominent mis-splicing in genes related to cardiac contractile function. Our study further reveals that the splicing regulation of RBPMS and RBPMS2 has the characteristics of "positional effects", i.e., the binding location on pre-mRNA dictates whether they function as splicing activators or repressors; and the intrinsic features of the target exon determine the requirement for one or two RBPMS proteins for splicing regulation. Our study sheds light on the functional importance of cardiac SF cooperation in maintaining cardiac splicing signature during heart development.
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Legere NJ, Hinson JT. Emerging CRISPR Therapies for Precision Gene Editing and Modulation in the Cardiovascular Clinic. Curr Cardiol Rep 2024; 26:1231-1240. [PMID: 39287778 DOI: 10.1007/s11886-024-02125-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 08/21/2024] [Indexed: 09/19/2024]
Abstract
PURPOSE OF REVIEW Outline the growing suite of novel genome editing tools powered by CRISPR-Cas9 technology that are rapidly advancing towards the clinic for the treatment of cardiovascular disorders. RECENT FINDINGS A diversity of new genome editors and modulators are being developed for therapies across myriad human diseases. Recent breakthroughs have improved the efficacy, safety, specificity, and delivery of CRISPR-mediated therapies that could impact heart disease in the next decade, though several challenges remain. Many iterations of the original CRISPR system have been developed seeking to leverage its vast therapeutic potential. As examples, nuclease-free editing, precision single-nucleotide editing, gene expression regulation, and epigenomic modifications are now feasible with the current CRISPR-mediated suite of enzymes. These emerging tools will be indispensable for the development of novel cardiovascular therapeutics as demonstrated by recent successes in both basic research laboratories and pre-clinical models. Here, we provide an overview of current and emerging CRISPR-mediated technologies as they pertain to the cardiovascular system, highlighting successful implementations and future challenges.
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Affiliation(s)
| | - J Travis Hinson
- University of Connecticut Health Center, Farmington, CT, USA.
- The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA.
- Calhoun Cardiology Center, UConn Health, Farmington, CT, USA.
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Wagner T, Priyanka P, Micheletti R, Friedman MJ, Nair SJ, Gamliel A, Taylor H, Song X, Cho M, Oh S, Li W, Han J, Ohgi KA, Abrass M, D'Antonio-Chronowska A, D'Antonio M, Hazuda H, Duggirala R, Blangero J, Ding S, Guzmann C, Frazer KA, Aggarwal AK, Zemljic-Harpf AE, Rosenfeld MG, Suh Y. Recruitment of CTCF to the SIRT1 promoter after Oxidative Stress mediates Cardioprotective Transcription. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.05.17.594600. [PMID: 38798402 PMCID: PMC11118446 DOI: 10.1101/2024.05.17.594600] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/29/2024]
Abstract
Because most DNA-binding transcription factors (dbTFs), including the architectural regulator CTCF, bind RNA and exhibit di-/multimerization, a central conundrum is whether these distinct properties are regulated post-transcriptionally to modulate transcriptional programs. Here, investigating stress-dependent activation of SIRT1, encoding an evolutionarily-conserved protein deacetylase, we show that induced phosphorylation of CTCF acts as a rheostat to permit CTCF occupancy of low-affinity promoter DNA sites to precisely the levels necessary. This CTCF recruitment to the SIRT1 promoter is eliciting a cardioprotective cardiomyocyte transcriptional activation program and provides resilience against the stress of the beating heart in vivo . Mice harboring a mutation in the conserved low-affinity CTCF promoter binding site exhibit an altered, cardiomyocyte-specific transcriptional program and a systolic heart failure phenotype. This transcriptional role for CTCF reveals that a covalent dbTF modification regulating signal-dependent transcription serves as a previously unsuspected component of the oxidative stress response.
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Bradford WH, Zhang J, Gutierrez-Lara EJ, Liang Y, Do A, Wang TM, Nguyen L, Mataraarachchi N, Wang J, Gu Y, McCulloch A, Peterson KL, Sheikh F. Plakophilin 2 gene therapy prevents and rescues arrhythmogenic right ventricular cardiomyopathy in a mouse model harboring patient genetics. NATURE CARDIOVASCULAR RESEARCH 2023; 2:1246-1261. [PMID: 39196150 PMCID: PMC11357983 DOI: 10.1038/s44161-023-00370-3] [Citation(s) in RCA: 22] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/23/2023] [Accepted: 10/16/2023] [Indexed: 08/29/2024]
Abstract
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a fatal genetic heart disease characterized by cardiac arrhythmias, in which fibrofatty deposition leads to heart failure, with no effective treatments. Plakophilin 2 (PKP2) is the most frequently mutated gene in ARVC, and although altered RNA splicing has been implicated, there are no models to study its effect and therapeutics. Here, we generate a mouse model harboring a PKP2 mutation (IVS10-1G>C) affecting RNA splicing, recapitulating ARVC features and sudden death starting at 4 weeks. Administering AAV-PKP2 gene therapy (adeno-associated viral therapy to drive cardiac expression of PKP2) to neonatal mice restored PKP2 protein levels, completely preventing cardiac desmosomal and pathological deficits associated with ARVC, ensuring 100% survival of mice up to 6 months. Late-stage AAV-PKP2 administration rescued desmosomal protein deficits and reduced pathological deficits including improved cardiac function in adult mice, resulting in 100% survival up to 4 months. We suggest that AAV-PKP2 gene therapy holds promise for circumventing ARVC associated with PKP2 mutations, including splice site mutations.
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Affiliation(s)
- William H Bradford
- Department of Medicine, University of California San Diego, La Jolla, CA, USA
| | - Jing Zhang
- Department of Medicine, University of California San Diego, La Jolla, CA, USA
| | | | - Yan Liang
- Department of Medicine, University of California San Diego, La Jolla, CA, USA
| | - Aryanne Do
- Department of Medicine, University of California San Diego, La Jolla, CA, USA
| | - Tsui-Min Wang
- Department of Medicine, University of California San Diego, La Jolla, CA, USA
| | - Lena Nguyen
- Department of Medicine, University of California San Diego, La Jolla, CA, USA
| | | | - Jie Wang
- Department of Medicine, University of California San Diego, La Jolla, CA, USA
| | - Yusu Gu
- Department of Medicine, University of California San Diego, La Jolla, CA, USA
| | - Andrew McCulloch
- Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
| | - Kirk L Peterson
- Department of Medicine, University of California San Diego, La Jolla, CA, USA
| | - Farah Sheikh
- Department of Medicine, University of California San Diego, La Jolla, CA, USA.
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Zhou X, Fang X, Ithychanda SS, Wu T, Gu Y, Chen C, Wang L, Bogomolovas J, Qin J, Chen J. Interaction of Filamin C With Actin Is Essential for Cardiac Development and Function. Circ Res 2023; 133:400-411. [PMID: 37492967 PMCID: PMC10529502 DOI: 10.1161/circresaha.123.322750] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/03/2023] [Accepted: 07/17/2023] [Indexed: 07/27/2023]
Abstract
BACKGROUND FLNC (filamin C), a member of the filamin family predominantly expressed in striated muscles, plays a crucial role in bridging the cytoskeleton and ECM (extracellular matrix) in cardiomyocytes, thereby maintaining heart integrity and function. Although genetic variants within the N-terminal ABD (actin-binding domain) of FLNC have been identified in patients with cardiomyopathy, the precise contribution of the actin-binding capability to FLNC's function in mammalian hearts remains poorly understood. METHODS We conducted in silico analysis of the 3-dimensional structure of mouse FLNC to identify key amino acid residues within the ABD that are essential for FLNC's actin-binding capacity. Subsequently, we performed coimmunoprecipitation and immunofluorescent assays to validate the in silico findings and assess the impact of these mutations on the interactions with other binding partners and the subcellular localization of FLNC. Additionally, we generated and analyzed knock-in mouse models in which the FLNC-actin interaction was completely disrupted by these mutations. RESULTS Our findings revealed that F93A/L98E mutations completely disrupted FLNC-actin interaction while preserving FLNC's ability to interact with other binding partners ITGB1 (β1 integrin) and γ-SAG (γ-sarcoglycan), as well as maintaining FLNC subcellular localization. Loss of FLNC-actin interaction in embryonic cardiomyocytes resulted in embryonic lethality and cardiac developmental defects, including ventricular wall malformation and reduced cardiomyocyte proliferation. Moreover, disruption of FLNC-actin interaction in adult cardiomyocytes led to severe dilated cardiomyopathy, enhanced lethality and dysregulation of key cytoskeleton components. CONCLUSIONS Our data strongly support the crucial role of FLNC as a bridge between actin filaments and ECM through its interactions with actin, ITGB1, γ-SAG, and other associated proteins in cardiomyocytes. Disruption of FLN-actin interaction may result in detachment of actin filaments from the extracellular matrix, ultimately impairing normal cardiac development and function. These findings also provide insights into mechanisms underlying cardiomyopathy associated with genetic variants in FLNC ABD and other regions.
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Affiliation(s)
- Xiaohai Zhou
- Department of Medicine (X.Z., X.F., T.W., Y.G., C.C., L.W., J.B., J.C.), University of California San Diego, La Jolla
| | - Xi Fang
- Department of Medicine (X.Z., X.F., T.W., Y.G., C.C., L.W., J.B., J.C.), University of California San Diego, La Jolla
| | - Sujay Subbayya Ithychanda
- Department of Cardiovascular and Metabolic Sciences (S.S.I., J.Q.), Lerner Research Institute, Cleveland Clinic, OH
| | - Tongbin Wu
- Department of Medicine (X.Z., X.F., T.W., Y.G., C.C., L.W., J.B., J.C.), University of California San Diego, La Jolla
| | - Yusu Gu
- Department of Medicine (X.Z., X.F., T.W., Y.G., C.C., L.W., J.B., J.C.), University of California San Diego, La Jolla
| | - Chao Chen
- Department of Medicine (X.Z., X.F., T.W., Y.G., C.C., L.W., J.B., J.C.), University of California San Diego, La Jolla
| | - Li Wang
- Department of Medicine (X.Z., X.F., T.W., Y.G., C.C., L.W., J.B., J.C.), University of California San Diego, La Jolla
| | - Julius Bogomolovas
- Department of Medicine (X.Z., X.F., T.W., Y.G., C.C., L.W., J.B., J.C.), University of California San Diego, La Jolla
| | - Jun Qin
- Department of Cardiovascular and Metabolic Sciences (S.S.I., J.Q.), Lerner Research Institute, Cleveland Clinic, OH
| | - Ju Chen
- Department of Medicine (X.Z., X.F., T.W., Y.G., C.C., L.W., J.B., J.C.), University of California San Diego, La Jolla
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Fixsen BR, Han CZ, Zhou Y, Spann NJ, Saisan P, Shen Z, Balak C, Sakai M, Cobo I, Holtman IR, Warden AS, Ramirez G, Collier JG, Pasillas MP, Yu M, Hu R, Li B, Belhocine S, Gosselin D, Coufal NG, Ren B, Glass CK. SALL1 enforces microglia-specific DNA binding and function of SMADs to establish microglia identity. Nat Immunol 2023; 24:1188-1199. [PMID: 37322178 PMCID: PMC10307637 DOI: 10.1038/s41590-023-01528-8] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2022] [Accepted: 05/04/2023] [Indexed: 06/17/2023]
Abstract
Spalt-like transcription factor 1 (SALL1) is a critical regulator of organogenesis and microglia identity. Here we demonstrate that disruption of a conserved microglia-specific super-enhancer interacting with the Sall1 promoter results in complete and specific loss of Sall1 expression in microglia. By determining the genomic binding sites of SALL1 and leveraging Sall1 enhancer knockout mice, we provide evidence for functional interactions between SALL1 and SMAD4 required for microglia-specific gene expression. SMAD4 binds directly to the Sall1 super-enhancer and is required for Sall1 expression, consistent with an evolutionarily conserved requirement of the TGFβ and SMAD homologs Dpp and Mad for cell-specific expression of Spalt in the Drosophila wing. Unexpectedly, SALL1 in turn promotes binding and function of SMAD4 at microglia-specific enhancers while simultaneously suppressing binding of SMAD4 to enhancers of genes that become inappropriately activated in enhancer knockout microglia, thereby enforcing microglia-specific functions of the TGFβ-SMAD signaling axis.
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Affiliation(s)
- Bethany R Fixsen
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
| | - Claudia Z Han
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
| | - Yi Zhou
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
| | - Nathanael J Spann
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
| | - Payam Saisan
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
| | - Zeyang Shen
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
| | - Christopher Balak
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
| | - Mashito Sakai
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
- Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan
| | - Isidoro Cobo
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
| | - Inge R Holtman
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
- Department of Biomedical Sciences of Cells and Systems, Section Molecular Neurobiology, University of Groningen and University Medical Center Groningen, Groningen, the Netherlands
| | - Anna S Warden
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
| | | | - Jana G Collier
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
| | - Martina P Pasillas
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
| | - Miao Yu
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
- Ludwig Institute for Cancer Research, La Jolla, CA, USA
| | - Rong Hu
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
- Ludwig Institute for Cancer Research, La Jolla, CA, USA
| | - Bin Li
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
- Ludwig Institute for Cancer Research, La Jolla, CA, USA
| | - Sarah Belhocine
- Axe Neuroscience, Centre de Recherche du CHU de Québec, Université Laval, Quebec, Quebec, Canada
- Département de Médecine Moléculaire de la Faculté de Médecine, Université Laval, Quebec, Quebec, Canada
| | - David Gosselin
- Axe Neuroscience, Centre de Recherche du CHU de Québec, Université Laval, Quebec, Quebec, Canada
- Département de Médecine Moléculaire de la Faculté de Médecine, Université Laval, Quebec, Quebec, Canada
| | - Nicole G Coufal
- Sanford Consortium for Regenerative Medicine, La Jolla, CA, USA
- Department of Pediatrics, School of Medicine, UC San Diego, La Jolla, CA, USA
| | - Bing Ren
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA
- Ludwig Institute for Cancer Research, La Jolla, CA, USA
| | - Christopher K Glass
- Department of Cellular and Molecular Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA.
- Department of Medicine, School of Medicine, UC San Diego, La Jolla, CA, USA.
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Polikarpova AV, Egorova TV, Lunev EA, Tsitrina AA, Vassilieva SG, Savchenko IM, Silaeva YY, Deykin AV, Bardina MV. CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics. Front Genome Ed 2023; 5:1034720. [PMID: 37077890 PMCID: PMC10106585 DOI: 10.3389/fgeed.2023.1034720] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2022] [Accepted: 03/20/2023] [Indexed: 04/05/2023] Open
Abstract
The development of personalized medicine for genetic diseases requires preclinical testing in the appropriate animal models. GNAO1 encephalopathy is a severe neurodevelopmental disorder caused by heterozygous de novo mutations in the GNAO1 gene. GNAO1 c.607 G>A is one of the most common pathogenic variants, and the mutant protein Gαo-G203R likely adversely affects neuronal signaling. As an innovative approach, sequence-specific RNA-based therapeutics such as antisense oligonucleotides or effectors of RNA interference are potentially applicable for selective suppression of the mutant GNAO1 transcript. While in vitro validation can be performed in patient-derived cells, a humanized mouse model to rule out the safety of RNA therapeutics is currently lacking. In the present work, we employed CRISPR/Cas9 technology to introduce a single-base substitution into exon 6 of the Gnao1 to replace the murine Gly203-coding triplet (GGG) with the codon used in the human gene (GGA). We verified that genome-editing did not interfere with the Gnao1 mRNA or Gαo protein synthesis and did not alter localization of the protein in the brain structures. The analysis of blastocysts revealed the off-target activity of the CRISPR/Cas9 complexes; however, no modifications of the predicted off-target sites were detected in the founder mouse. Histological staining confirmed the absence of abnormal changes in the brain of genome-edited mice. The created mouse model with the “humanized” fragment of the endogenous Gnao1 is suitable to rule out unintended targeting of the wild-type allele by RNA therapeutics directed at lowering GNAO1 c.607 G>A transcripts.
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Affiliation(s)
- Anna V. Polikarpova
- Laboratory of Modeling and Gene Therapy of Hereditary Diseases, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia
- Marlin Biotech, Sochi, Russia
| | - Tatiana V. Egorova
- Laboratory of Modeling and Gene Therapy of Hereditary Diseases, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia
- Marlin Biotech, Sochi, Russia
| | - Evgenii A. Lunev
- Laboratory of Modeling and Gene Therapy of Hereditary Diseases, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia
- Marlin Biotech, Sochi, Russia
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia
| | - Alexandra A. Tsitrina
- Koltzov Institute of Developmental Biology Russian Academy of Sciences, Moscow, Russia
| | - Svetlana G. Vassilieva
- Laboratory of Modeling and Gene Therapy of Hereditary Diseases, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia
- Marlin Biotech, Sochi, Russia
| | - Irina M. Savchenko
- Marlin Biotech, Sochi, Russia
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia
| | - Yuliya Y. Silaeva
- Core Facility Center, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia
| | - Alexey V. Deykin
- Marlin Biotech, Sochi, Russia
- Core Facility Center, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia
- Laboratory of Genetic Technologies and Genome Editing for Biomedicine and Animal Health, Joint Center for Genetic Technologies, Belgorod National Research University, Belgorod, Russia
| | - Maryana V. Bardina
- Laboratory of Modeling and Gene Therapy of Hereditary Diseases, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia
- Marlin Biotech, Sochi, Russia
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia
- *Correspondence: Maryana V. Bardina,
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Mollashahi B, Latifi-Navid H, Owliaee I, Shamdani S, Uzan G, Jamehdor S, Naserian S. Research and Therapeutic Approaches in Stem Cell Genome Editing by CRISPR Toolkit. Molecules 2023; 28:1982. [PMID: 36838970 PMCID: PMC9961668 DOI: 10.3390/molecules28041982] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2022] [Revised: 02/03/2023] [Accepted: 02/06/2023] [Indexed: 02/22/2023] Open
Abstract
The most widely used genome editing toolkit is CRISPR (clustered regularly interspaced short palindromic repeats). It provides the possibility of replacing and modifying DNA and RNA nucleotides. Furthermore, with advancements in biological technology, inhibition and activation of the transcription of specific gene(s) has become possible. Bioinformatics tools that target the evolution of CRISPR-associated protein 9 (Cas9) turn this protein into a vehicle that is specific for a DNA or RNA region with single guide RNA (sgRNA). This toolkit could be used by researchers to investigate the function of stem cell gene(s). Here, in this review article, we cover recent developments and applications of this technique in stem cells for research and clinical purposes and discuss different CRISPR/Cas technologies for knock-out, knock-in, activation, or inhibition of gene expression. Additionally, a comparison of several deliveries and off-target detecting strategies is discussed.
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Affiliation(s)
- Behrouz Mollashahi
- Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, 40126 Bologna, Italy
| | - Hamid Latifi-Navid
- Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran 14965/161, Iran
| | - Iman Owliaee
- Department of Virology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamedan 6517838636, Iran
| | - Sara Shamdani
- INSERM UMR-S-MD 1197, Hôpital Paul Brousse, Paris-Saclay University, 94807 Villejuif, France
- CellMedEx, 94100 Saint Maur Des Fossés, France
| | - Georges Uzan
- INSERM UMR-S-MD 1197, Hôpital Paul Brousse, Paris-Saclay University, 94807 Villejuif, France
| | - Saleh Jamehdor
- Department of Virology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamedan 6517838636, Iran
| | - Sina Naserian
- INSERM UMR-S-MD 1197, Hôpital Paul Brousse, Paris-Saclay University, 94807 Villejuif, France
- CellMedEx, 94100 Saint Maur Des Fossés, France
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Abstract
Generation of conditional knockout mice using the Cre-loxP system is essential for the analysis of gene functions. The use of CRISPR-Cas9 in combination with two sets of guide RNAs and single-stranded oligonucleotides including loxP sites enables simultaneous insertion of two loxP sequences. Unfortunately, this method induces double-strand breaks at two sites in the same chromosome, which causes an undesirable large chromosomal deletion and reduces the flanked loxP (flox) rate. To overcome this problem, we have developed a method that sequentially introduces each loxP sequence by electroporation at the one- and two-cell embryonic stages, respectively. This sequential electroporation method improves the floxing efficiency compared with the conventional simultaneous method, leading to a high yield of offspring with floxed alleles.
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Affiliation(s)
- Takuro Horii
- Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, Japan.
| | - Ryosuke Kobayashi
- Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, Japan
| | - Izuho Hatada
- Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, Japan. .,Viral Vector Core, Gunma University Initiative for Advanced Research (GIAR), Maebashi, Gunma, Japan.
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11
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Wei J, Guo W, Wang R, Paul Estillore J, Belke D, Chen YX, Vallmitjana A, Benitez R, Hove-Madsen L, Chen SRW. RyR2 Serine-2030 PKA Site Governs Ca 2+ Release Termination and Ca 2+ Alternans. Circ Res 2023; 132:e59-e77. [PMID: 36583384 DOI: 10.1161/circresaha.122.321177] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
BACKGROUND PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca2+ release and induction of cardiac Ca2+ alternans. METHODS We performed single-cell endoplasmic reticulum Ca2+ imaging to assess the impact of S2030 mutations on Ca2+ release termination in human embryonic kidney 293 cells. Here we determined the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation and carried out confocal Ca2+ imaging. RESULTS We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca2+ level at which Ca2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM-wild-type increased, whereas Ca2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca2+/CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM-wild-type, CaM-M, and PKA, but not CaMKII, in Ca2+ release termination. Moreover, we showed that isoproterenol and CaM-M suppressed pacing-induced Ca2+ alternans and accelerated Ca2+ transient recovery in intact working hearts, whereas CaM-wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide and the CaMKII inhibitor N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide individually and together, respectively. S2030L abolished the impact of CaM-wild-type, CaM-M, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide-sensitive component, but not the N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide-sensitive component, of isoproterenol.
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Affiliation(s)
- Jinhong Wei
- Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, Alberta T2N 4N1, Canada (J.W., W.G., R.W., J.P.E., D.B., Y.-X.C., S.R.W.C.).,School of Medicine, Northwest University, Xi 'an, China (J.W.)
| | - Wenting Guo
- Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, Alberta T2N 4N1, Canada (J.W., W.G., R.W., J.P.E., D.B., Y.-X.C., S.R.W.C.)
| | - Ruiwu Wang
- Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, Alberta T2N 4N1, Canada (J.W., W.G., R.W., J.P.E., D.B., Y.-X.C., S.R.W.C.)
| | - John Paul Estillore
- Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, Alberta T2N 4N1, Canada (J.W., W.G., R.W., J.P.E., D.B., Y.-X.C., S.R.W.C.)
| | - Darrell Belke
- Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, Alberta T2N 4N1, Canada (J.W., W.G., R.W., J.P.E., D.B., Y.-X.C., S.R.W.C.)
| | - Yong-Xiang Chen
- Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, Alberta T2N 4N1, Canada (J.W., W.G., R.W., J.P.E., D.B., Y.-X.C., S.R.W.C.)
| | | | - Raul Benitez
- Department of Automatic Control, Universitat Politècnica de Catalunya, 08034 Barcelona, Spain (A.V., R.B.)
| | - Leif Hove-Madsen
- Biomedical Research Institute Barcelona IIBB-CSIC, IIB Sant Pau and CIBERCV, Hospital de Sant Pau, 08025, Barcelona, Spain (L.H.-M.)
| | - S R Wayne Chen
- Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, Alberta T2N 4N1, Canada (J.W., W.G., R.W., J.P.E., D.B., Y.-X.C., S.R.W.C.)
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12
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Brian BF, Sauer ML, Greene JT, Senevirathne SE, Lindstedt AJ, Funk OL, Ruis BL, Ramirez LA, Auger JL, Swanson WL, Nunez MG, Moriarity BS, Lowell CA, Binstadt BA, Freedman TS. A dominant function of LynB kinase in preventing autoimmunity. SCIENCE ADVANCES 2022; 8:eabj5227. [PMID: 35452291 PMCID: PMC9032976 DOI: 10.1126/sciadv.abj5227] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/18/2021] [Accepted: 03/08/2022] [Indexed: 06/14/2023]
Abstract
Here, we report that the LynB splice variant of the Src-family kinase Lyn exerts a dominant immunosuppressive function in vivo, whereas the LynA isoform is uniquely required to restrain autoimmunity in female mice. We used CRISPR-Cas9 gene editing to constrain lyn splicing and expression, generating single-isoform LynA knockout (LynAKO) or LynBKO mice. Autoimmune disease in total LynKO mice is characterized by production of antinuclear antibodies, glomerulonephritis, impaired B cell development, and overabundance of activated B cells and proinflammatory myeloid cells. Expression of LynA or LynB alone uncoupled the developmental phenotype from the autoimmune disease: B cell transitional populations were restored, but myeloid cells and differentiated B cells were dysregulated. These changes were isoform-specific, sexually dimorphic, and distinct from the complete LynKO. Despite the apparent differences in disease etiology and penetrance, loss of either LynA or LynB had the potential to induce severe autoimmune disease with parallels to human systemic lupus erythematosus (SLE).
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Affiliation(s)
- Ben F. Brian
- Graduate Program in Molecular Pharmacology and Therapeutics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Monica L. Sauer
- Graduate Program in Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Joseph T. Greene
- Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA
| | - S. Erandika Senevirathne
- Graduate Program in Molecular Pharmacology and Therapeutics, University of Minnesota, Minneapolis, MN 55455, USA
| | - Anders J. Lindstedt
- Graduate Program in Microbiology, Immunology, and Cancer Biology, University of Minnesota, Minneapolis, MN 55455, USA
- Medical Scientist Training Program, University of Minnesota, Minneapolis, MN 55455, USA
| | - Olivia L. Funk
- Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA
| | - Brian L. Ruis
- Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
| | - Luis A. Ramirez
- Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA
| | - Jennifer L. Auger
- Department of Pediatrics, Division of Rheumatology, Allergy and Immunology, University of Minnesota, Minneapolis, MN 55455, USA
| | - Whitney L. Swanson
- Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA
| | - Myra G. Nunez
- Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA
| | - Branden S. Moriarity
- Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
- Division of Pediatric Hematology and Oncology, Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
- Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
| | - Clifford A. Lowell
- Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Bryce A. Binstadt
- Department of Pediatrics, Division of Rheumatology, Allergy and Immunology, University of Minnesota, Minneapolis, MN 55455, USA
- Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA
| | - Tanya S. Freedman
- Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
- Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA
- Center for Autoimmune Diseases Research, University of Minnesota, Minneapolis, MN 55455, USA
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13
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Sentmanat MF, White JM, Kouranova E, Cui X. Highly reliable creation of floxed alleles by electroporating single-cell embryos. BMC Biol 2022; 20:31. [PMID: 35115009 PMCID: PMC8815186 DOI: 10.1186/s12915-021-01223-w] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2021] [Accepted: 12/24/2021] [Indexed: 12/18/2022] Open
Abstract
BACKGROUND Floxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. However, an efficient and reliable strategy to flox genomic regions of any desired size is still lacking. RESULTS Here, we demonstrate that the method combining electroporation of fertilized eggs with gRNA/Cas9 complexes and single-stranded oligodeoxynucleotides (ssODNs), assessing phasing of loxP insertions in founders using an in vitro Cre assay and an optional, highly specific and efficient second-round targeting ensures the generation of floxed F1 animals in roughly five months for a wide range of sequence lengths (448 bp to 160 kb reported here). CONCLUSIONS Floxed alleles can be reliably obtained in a predictable timeline using the improved method of electroporation of two gRNA/Cas9 ribonucleoprotein particles (RNPs) and two ssODNs.
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Affiliation(s)
- Monica F. Sentmanat
- Genome Engineering & Stem Cell Center (GESC@MGI), Department of Genetics, Washington University in St. Louis School of Medicine, 660 S. Euclid Ave, St. Louis, MO 63110 USA
| | - J. Michael White
- Transgenic, Knockout and Microinjection Core, Department of Pathology & Immunology, Washington University in St. Louis School of Medicine, 660 S. Euclid Ave, St. Louis, MO 63110 USA
| | - Evguenia Kouranova
- Genome Engineering & Stem Cell Center (GESC@MGI), Department of Genetics, Washington University in St. Louis School of Medicine, 660 S. Euclid Ave, St. Louis, MO 63110 USA
| | - Xiaoxia Cui
- Genome Engineering & Stem Cell Center (GESC@MGI), Department of Genetics, Washington University in St. Louis School of Medicine, 660 S. Euclid Ave, St. Louis, MO 63110 USA
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14
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Bang ML, Bogomolovas J, Chen J. Understanding the molecular basis of cardiomyopathy. Am J Physiol Heart Circ Physiol 2022; 322:H181-H233. [PMID: 34797172 PMCID: PMC8759964 DOI: 10.1152/ajpheart.00562.2021] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/13/2021] [Revised: 11/16/2021] [Accepted: 11/16/2021] [Indexed: 02/03/2023]
Abstract
Inherited cardiomyopathies are a major cause of mortality and morbidity worldwide and can be caused by mutations in a wide range of proteins located in different cellular compartments. The present review is based on Dr. Ju Chen's 2021 Robert M. Berne Distinguished Lectureship of the American Physiological Society Cardiovascular Section, in which he provided an overview of the current knowledge on the cardiomyopathy-associated proteins that have been studied in his laboratory. The review provides a general summary of the proteins in different compartments of cardiomyocytes associated with cardiomyopathies, with specific focus on the proteins that have been studied in Dr. Chen's laboratory.
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Affiliation(s)
- Marie-Louise Bang
- Institute of Genetic and Biomedical Research (IRGB), National Research Council (CNR), Milan Unit, Milan, Italy
- IRCCS Humanitas Research Hospital, Rozzano (Milan), Italy
| | - Julius Bogomolovas
- Division of Cardiovascular Medicine, Department of Medicine Cardiology, University of California, San Diego, La Jolla, California
| | - Ju Chen
- Division of Cardiovascular Medicine, Department of Medicine Cardiology, University of California, San Diego, La Jolla, California
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15
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Xu M, Weng Q, Ji J. Applications and advances of CRISPR/Cas9 in animal cancer model. Brief Funct Genomics 2021; 19:235-241. [PMID: 32124927 DOI: 10.1093/bfgp/elaa002] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2019] [Revised: 01/07/2020] [Indexed: 01/18/2023] Open
Abstract
The recent developments of clustered regularly interspaced short palindromic repeats(CRISPR)/-associate protein 9 (CRISPR/Cas9) have got scientific interests due to the straightforward, efficient and versatile talents of it. Furthermore, the CRISPR/Cas9 system has democratized access to gene editing in many biological fields, including cancer. Cancer development is a multistep process caused by innate and acquired mutations and leads to the initiation and progression of tumorigenesis. It is obvious that establishing appropriate animal cancer models which can simulate human cancers is crucial for cancer research currently. Since the emergence of CRISPR/Cas9, considerable efforts have been taken by researchers to apply this technology in generating animal cancer models. Although there is still a long way to go we are happy to see the achievements we have made and the promising future we have.
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16
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Liang Y, Lyon RC, Pellman J, Bradford WH, Lange S, Bogomolovas J, Dalton ND, Gu Y, Bobar M, Lee MH, Iwakuma T, Nigam V, Asimaki A, Scheinman M, Peterson KL, Sheikh F. Desmosomal COP9 regulates proteome degradation in arrhythmogenic right ventricular dysplasia/cardiomyopathy. J Clin Invest 2021; 131:137689. [PMID: 33857019 PMCID: PMC8159691 DOI: 10.1172/jci137689] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2020] [Accepted: 04/14/2021] [Indexed: 12/28/2022] Open
Abstract
Dysregulated protein degradative pathways are increasingly recognized as mediators of human disease. This mechanism may have particular relevance to desmosomal proteins that play critical structural roles in both tissue architecture and cell-cell communication, as destabilization/breakdown of the desmosomal proteome is a hallmark of genetic-based desmosomal-targeted diseases, such as the cardiac disease arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). However, no information exists on whether there are resident proteins that regulate desmosomal proteome homeostasis. Here, we uncovered a cardiac constitutive photomorphogenesis 9 (COP9) desmosomal resident protein complex, composed of subunit 6 of the COP9 signalosome (CSN6), that enzymatically restricted neddylation and targeted desmosomal proteome degradation. CSN6 binding, localization, levels, and function were affected in hearts of classic mouse and human models of ARVD/C affected by desmosomal loss and mutations, respectively. Loss of desmosomal proteome degradation control due to junctional reduction/loss of CSN6 and human desmosomal mutations destabilizing junctional CSN6 were also sufficient to trigger ARVD/C in mice. We identified a desmosomal resident regulatory complex that restricted desmosomal proteome degradation and disease.
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Affiliation(s)
- Yan Liang
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - Robert C. Lyon
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - Jason Pellman
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - William H. Bradford
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - Stephan Lange
- Department of Medicine, University of California San Diego, La Jolla, California, USA
- Institute of Medicine, Department of Molecular and Clinical Medicine and Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden
| | - Julius Bogomolovas
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - Nancy D. Dalton
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - Yusu Gu
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - Marcus Bobar
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - Mong-Hong Lee
- Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Tomoo Iwakuma
- Department of Cancer Biology, University of Kansas Medical Center, Kansas City, Kansas, USA
| | - Vishal Nigam
- Department of Pediatrics, University of California San Diego, La Jolla, California, USA
- Department of Pediatrics, Seattle Children’s Research Institute and University of Washington, Seattle, Washington, USA
| | - Angeliki Asimaki
- Cardiology Clinical Academic Group, St. George’s University of London, London, United Kingdom
| | - Melvin Scheinman
- Department of Medicine, Cardiac Electrophysiology Section, University of California San Francisco, San Francisco, California, USA
| | - Kirk L. Peterson
- Department of Medicine, University of California San Diego, La Jolla, California, USA
| | - Farah Sheikh
- Department of Medicine, University of California San Diego, La Jolla, California, USA
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17
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Morin F, Singh N, Mdzomba JB, Dumas A, Pernet V, Vallières L. Conditional Deletions of Hdc Confirm Roles of Histamine in Anaphylaxis and Circadian Activity but Not in Autoimmune Encephalomyelitis. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2021; 206:2029-2037. [PMID: 33846226 DOI: 10.4049/jimmunol.2000719] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/17/2020] [Accepted: 03/01/2021] [Indexed: 12/12/2022]
Abstract
Histamine is best known for its role in allergies, but it could also be involved in autoimmune diseases such as multiple sclerosis. However, studies using experimental autoimmune encephalomyelitis (EAE), the most widely used animal model for multiple sclerosis, have reported conflicting observations and suggest the implication of a nonclassical source of histamine. In this study, we demonstrate that neutrophils are the main producers of histamine in the spinal cord of EAE mice. To assess the role of histamine by taking into account its different cellular sources, we used CRISPR-Cas9 to generate conditional knockout mice for the histamine-synthesizing enzyme histidine decarboxylase. We found that ubiquitous and cell-specific deletions do not affect the course of EAE. However, neutrophil-specific deletion attenuates hypothermia caused by IgE-mediated anaphylaxis, whereas neuron-specific deletion reduces circadian activity. In summary, this study refutes the role of histamine in EAE, unveils a role for neutrophil-derived histamine in IgE-mediated anaphylaxis, and establishes a new mouse model to re-explore the inflammatory and neurologic roles of histamine.
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MESH Headings
- Anaphylaxis/genetics
- Anaphylaxis/immunology
- Anaphylaxis/metabolism
- Animals
- Cells, Cultured
- Circadian Rhythm/immunology
- Disease Models, Animal
- Encephalomyelitis, Autoimmune, Experimental/genetics
- Encephalomyelitis, Autoimmune, Experimental/immunology
- Encephalomyelitis, Autoimmune, Experimental/metabolism
- Histamine/immunology
- Histamine/metabolism
- Histidine Decarboxylase/genetics
- Histidine Decarboxylase/immunology
- Histidine Decarboxylase/metabolism
- Humans
- Kaplan-Meier Estimate
- Male
- Mice, Inbred C57BL
- Mice, Knockout
- Mice, Transgenic
- Multiple Sclerosis/genetics
- Multiple Sclerosis/immunology
- Multiple Sclerosis/metabolism
- Neutrophils/cytology
- Neutrophils/immunology
- Neutrophils/metabolism
- Spinal Cord/immunology
- Spinal Cord/metabolism
- Mice
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Affiliation(s)
- Françoise Morin
- Neuroscience Unit, University Hospital Center of Quebec, Laval University, Quebec City, Quebec, Canada
| | - Noopur Singh
- Neuroscience Unit, University Hospital Center of Quebec, Laval University, Quebec City, Quebec, Canada
| | - Julius Baya Mdzomba
- Regenerative Medicine Unit, University Hospital Center of Quebec, Laval University, Quebec City, Quebec, Canada
| | - Aline Dumas
- Neuroscience Unit, University Hospital Center of Quebec, Laval University, Quebec City, Quebec, Canada
| | - Vincent Pernet
- Regenerative Medicine Unit, University Hospital Center of Quebec, Laval University, Quebec City, Quebec, Canada
- Department of Neurology, Inselspital Bern, University Hospital, University of Bern, Bern, Switzerland
| | - Luc Vallières
- Neuroscience Unit, University Hospital Center of Quebec, Laval University, Quebec City, Quebec, Canada;
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18
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Yang H, Wang H, Jaenisch R. Response to "Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation". Genome Biol 2021; 22:98. [PMID: 33827646 PMCID: PMC8025483 DOI: 10.1186/s13059-021-02312-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Affiliation(s)
- Hui Yang
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.
| | - Haoyi Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing, China.
| | - Rudolf Jaenisch
- Whitehead Institute for Biomedical Research, Cambridge, MA, USA. .,Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
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19
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Liu C, Spinozzi S, Feng W, Chen Z, Zhang L, Zhu S, Wu T, Fang X, Ouyang K, Evans SM, Chen J. Homozygous G650del nexilin variant causes cardiomyopathy in mice. JCI Insight 2020; 5:138780. [PMID: 32814711 PMCID: PMC7455123 DOI: 10.1172/jci.insight.138780] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2020] [Accepted: 07/09/2020] [Indexed: 01/28/2023] Open
Abstract
Nexilin (NEXN) was recently identified as a component of the junctional membrane complex required for development and maintenance of cardiac T-tubules. Loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy (DCM) and premature death. A 3 bp deletion (1948-1950del) leading to loss of the glycine in position 650 (G650del) is classified as a variant of uncertain significance in humans and may function as an intermediate risk allele. To determine the effect of the G650del variant on cardiac structure and function, we generated a G645del-knockin (G645del is equivalent to human G650del) mouse model. Homozygous G645del mice express about 30% of the Nexn expressed by WT controls and exhibited a progressive DCM characterized by reduced T-tubule formation, with disorganization of the transverse-axial tubular system. On the other hand, heterozygous Nexn global KO mice and genetically engineered mice encoding a truncated Nexn missing the first N-terminal actin-binding domain exhibited normal cardiac function, despite expressing only 50% and 20% of the Nexn, respectively, expressed by WT controls, suggesting that not only quantity but also quality of Nexn is necessary for a proper function. These findings demonstrated that Nexn G645 is crucial for Nexn's function in tubular system organization and normal cardiac function.
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Affiliation(s)
- Canzhao Liu
- Department of Medicine, UCSD, La Jolla, California, USA
| | | | - Wei Feng
- Department of Medicine, UCSD, La Jolla, California, USA
| | - Ze’e Chen
- Department of Medicine, UCSD, La Jolla, California, USA
- Drug Discovery Center, State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China
| | - Lunfeng Zhang
- Department of Medicine, UCSD, La Jolla, California, USA
- Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, UCSD, La Jolla, California, USA
| | - Siting Zhu
- Department of Medicine, UCSD, La Jolla, California, USA
- Drug Discovery Center, State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China
| | - Tongbin Wu
- Department of Medicine, UCSD, La Jolla, California, USA
| | - Xi Fang
- Department of Medicine, UCSD, La Jolla, California, USA
| | - Kunfu Ouyang
- Drug Discovery Center, State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China
| | - Sylvia M. Evans
- Department of Medicine, UCSD, La Jolla, California, USA
- Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, UCSD, La Jolla, California, USA
| | - Ju Chen
- Department of Medicine, UCSD, La Jolla, California, USA
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20
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Nakamura T, Morishige S, Ozawa H, Kuboyama K, Yamasaki Y, Oya S, Yamaguchi M, Aoyama K, Seki R, Mouri F, Osaki K, Okamura T, Mizuno S, Nagafuji K. Successful correction of factor V deficiency of patient-derived iPSCs by CRISPR/Cas9-mediated gene editing. Haemophilia 2020; 26:826-833. [PMID: 32700411 DOI: 10.1111/hae.14104] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2020] [Revised: 05/07/2020] [Accepted: 06/22/2020] [Indexed: 12/21/2022]
Abstract
BACKGROUND Factor V (FV) deficiency is a monogenic inherited coagulation disorder considered to be an ideal indication for gene therapy. To investigate the possibility of therapeutic application of genome editing, we generated induced pluripotent stem cells (iPSCs) from a FV-deficient patient and repaired the mutation of factor V gene (F5) using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9). METHODS The patient's peripheral blood mononuclear cells were reprogrammed for iPSCs. The targeting vector was designed with homology arms against F5 containing the corrected sequence. Cas9 ribonucleoprotein (RNP) complex and targeting vector were electroporated into iPSCs. Gene-edited iPSCs were differentiated into hepatocyte-like cells (HLCs). RESULTS The mutation of F5 in patient-derived iPSCs was repaired by CRISPR/Cas9. In concentrated culture supernatants of patient-derived iPS-HLCs, neither FV antigen nor activity was detected, while in those of gene-corrected iPS-HLCs, FV antigen and specific activity were 67.0 ± 13.1 ng/mL and 173.2 ± 41.1 U/mg, respectively. CONCLUSIONS We successfully repaired the mutation of F5 using the CRISPR/Cas9 and confirmed the recovery of FV activity with gene-corrected iPS-HLCs. Gene-edited iPSCs are promising for elucidating the pathophysiology as well as for a modality of gene therapy.
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Affiliation(s)
- Takayuki Nakamura
- Division of Hematology and Oncology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan
| | - Satoshi Morishige
- Division of Hematology and Oncology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan
| | - Hidetoshi Ozawa
- Division of Hematology and Oncology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan
| | - Kenji Kuboyama
- Department of Clinical Laboratory Medicine, Kurume University Hospital, Kurume, Japan
| | - Yoshitaka Yamasaki
- Division of Hematology and Oncology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan
| | - Shuki Oya
- Division of Hematology and Oncology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan
| | - Maki Yamaguchi
- Division of Hematology and Oncology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan
| | - Kazutoshi Aoyama
- Division of Hematology and Oncology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan
| | - Ritsuko Seki
- Division of Hematology and Oncology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan
| | - Fumihiko Mouri
- Division of Hematology and Oncology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan
| | - Koichi Osaki
- Division of Hematology and Oncology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan
| | - Takashi Okamura
- Division of Hematology and Oncology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan.,Center for Hematology and Oncology, St. Mary's Hospital, Kurume, Japan
| | - Shinichi Mizuno
- Division of Hematology and Oncology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan.,Division of Medical Sciences and Technology, Department of Health Sciences, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Koji Nagafuji
- Division of Hematology and Oncology, Department of Medicine, Kurume University School of Medicine, Kurume, Japan
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21
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Bogomolovas J, Feng W, Yu MD, Huang S, Zhang L, Trexler C, Gu Y, Spinozzi S, Chen J. Atypical ALPK2 kinase is not essential for cardiac development and function. Am J Physiol Heart Circ Physiol 2020; 318:H1509-H1515. [PMID: 32383995 PMCID: PMC7311700 DOI: 10.1152/ajpheart.00249.2020] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/10/2020] [Revised: 04/28/2020] [Accepted: 05/05/2020] [Indexed: 01/18/2023]
Abstract
Protein kinases play an integral role in cardiac development, function, and disease. Recent experimental and clinical data have implied that protein kinases belonging to a family of atypical α-protein kinases, including α-protein kinase 2 (ALPK2), are important for regulating cardiac development and maintaining function via regulation of WNT signaling. A recent study in zebrafish reported that loss of ALPK2 leads to severe cardiac defects; however, the relevance of ALPK2 has not been studied in a mammalian animal model. To assess the role of ALPK2 in the mammalian heart, we generated two independent global Alpk2-knockout (Alpk2-gKO) mouse lines, using CRISPR/Cas9 technology. We performed physiological and biochemical analyses of Alpk2-gKO mice to determine the functional, morphological, and molecular consequences of Alpk2 deletion at the organismal level. We found that Alpk2-gKO mice exhibited normal cardiac function and morphology up to one year of age. Moreover, we did not observe altered WNT signaling in neonatal Alpk2-gKO mouse hearts. In conclusion, Alpk2 is dispensable for cardiac development and function in the murine model. Our results suggest that Alpk2 is a rapidly evolving gene that lost its essential cardiac functions in mammals.NEW & NOTEWORTHY Several studies indicated the importance of ALPK2 for cardiac function and development. A recent study in zebrafish report that loss of ALPK2 leads to severe cardiac defects. In contrast, murine Alpk2-gKO models developed in this work display no overt cardiac phenotype. Our results suggest ALPK2, as a rapidly evolving gene, lost its essential cardiac functions in mammals.
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Affiliation(s)
- Julius Bogomolovas
- Department of Medicine, University of California, San Diego, La Jolla, California
| | - Wei Feng
- Department of Medicine, University of California, San Diego, La Jolla, California
| | - Matthew Daniel Yu
- Department of Medicine, University of California, San Diego, La Jolla, California
| | - Serena Huang
- Department of Medicine, University of California, San Diego, La Jolla, California
| | - Lunfeng Zhang
- Department of Medicine, University of California, San Diego, La Jolla, California
| | - Christa Trexler
- Department of Medicine, University of California, San Diego, La Jolla, California
| | - Yusu Gu
- Department of Medicine, University of California, San Diego, La Jolla, California
| | - Simone Spinozzi
- Department of Medicine, University of California, San Diego, La Jolla, California
| | - Ju Chen
- Department of Medicine, University of California, San Diego, La Jolla, California
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22
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Horii T, Kobayashi R, Kimura M, Morita S, Hatada I. Calcium-Free and Cytochalasin B Treatment Inhibits Blastomere Fusion in 2-Cell Stage Embryos for the Generation of Floxed Mice via Sequential Electroporation. Cells 2020; 9:cells9051088. [PMID: 32354036 PMCID: PMC7290713 DOI: 10.3390/cells9051088] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2020] [Revised: 04/24/2020] [Accepted: 04/27/2020] [Indexed: 01/10/2023] Open
Abstract
The generation of conditional knockout mice using the Cre-loxP system is advantageous for the functional analysis of genes. Flanked by two loxP sites (floxed) mice can be directly obtained from fertilized eggs by the CRISPR/Cas9 genome editing system. We previously reported that sequential knock-in (KI) of each loxP site by electroporation (EP) at the 1- and 2-cell embryonic stages increases the number of mice with floxed alleles compared with simultaneous KI. However, EP at the 2-cell stage frequently induced blastomere fusion. These fused embryos cannot develop to term because they are tetraploidized. In this study, we examined the following three conditions to inhibit blastomere fusion by EP at the 2-cell stage: (1) hypertonic treatment, (2) Calcium (Ca2+)-free treatment, and (3) actin polymerization inhibition. Hypertonic treatment of 2-cell stage embryos prevented blastomere fusion and facilitated blastocyst development; however, KI efficiency was decreased. Ca2+-free treatment and actin polymerization inhibition by cytochalasin B (CB) reduced fusion rate, and did not have negative effects on development and KI efficiency. These results suggest that Ca2+-free and CB treatment at the 2-cell stage is effective to generate floxed mice in combination with a sequential EP method.
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23
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Shimizu A, Zankov DP, Sato A, Komeno M, Toyoda F, Yamazaki S, Makita T, Noda T, Ikawa M, Asano Y, Miyashita Y, Takashima S, Morita H, Ishikawa T, Makita N, Hitosugi M, Matsuura H, Ohno S, Horie M, Ogita H. Identification of transmembrane protein 168 mutation in familial Brugada syndrome. FASEB J 2020; 34:6399-6417. [PMID: 32175648 DOI: 10.1096/fj.201902991r] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2019] [Revised: 02/19/2020] [Accepted: 03/02/2020] [Indexed: 12/30/2022]
Abstract
Brugada syndrome (BrS) is an inherited channelopathy responsible for almost 20% of sudden cardiac deaths in patients with nonstructural cardiac diseases. Approximately 70% of BrS patients, the causative gene mutation(s) remains unknown. In this study, we used whole exome sequencing to investigate candidate mutations in a family clinically diagnosed with BrS. A heterozygous 1616G>A substitution (R539Q mutation) was identified in the transmembrane protein 168 (TMEM168) gene of symptomatic individuals. Similar to endogenous TMEM168, both TMEM168 wild-type (WT) and mutant proteins that were ectopically induced in HL-1 cells showed nuclear membrane localization. A significant decrease in Na+ current and Nav 1.5 protein expression was observed in HL-1 cardiomyocytes expressing mutant TMEM168. Ventricular tachyarrhythmias and conduction disorders were induced in the heterozygous Tmem168 1616G>A knock-in mice by pharmacological stimulation, but not in WT mice. Na+ current was reduced in ventricular cardiomyocytes isolated from the Tmem168 knock-in heart, and Nav 1.5 expression was also impaired. This impairment was dependent on increased Nedd4-2 binding to Nav 1.5 and subsequent ubiquitination. Collectively, our results show an association between the TMEM168 1616G>A mutation and arrhythmogenesis in a family with BrS.
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Affiliation(s)
- Akio Shimizu
- Division of Molecular Medical Biochemistry, Department of Biochemistry and Molecular Biology, Shiga University of Medical Science, Otsu, Japan
| | - Dimitar P Zankov
- Division of Molecular Medical Biochemistry, Department of Biochemistry and Molecular Biology, Shiga University of Medical Science, Otsu, Japan.,Department of Bioscience and Genetics, National Cerebral and Cardiovascular Center, Suita, Japan
| | - Akira Sato
- Division of Molecular Medical Biochemistry, Department of Biochemistry and Molecular Biology, Shiga University of Medical Science, Otsu, Japan
| | - Masahiro Komeno
- Division of Molecular Medical Biochemistry, Department of Biochemistry and Molecular Biology, Shiga University of Medical Science, Otsu, Japan
| | - Futoshi Toyoda
- Division of Cell Physiology, Department of Physiology, Shiga University of Medical Science, Otsu, Japan
| | - Satoru Yamazaki
- Department of Molecular Pharmacology, National Cerebral and Cardiovascular Center, Suita, Japan
| | - Toshinori Makita
- Division of Cardiac Electrophysiology, Department of Cardiovascular Center, Osaka Red Cross Hospital, Osaka, Japan
| | - Taichi Noda
- Animal Resource Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Japan
| | - Masahito Ikawa
- Animal Resource Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Japan
| | - Yoshihiro Asano
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Japan
| | - Yohei Miyashita
- Department of Legal Medicine, Osaka University Graduate School of Medicine, Suita, Japan
| | - Seiji Takashima
- Department of Medical Biochemistry, Osaka University Graduate School of Medicine, Suita, Japan
| | - Hiroshi Morita
- Department of Cardiovascular Therapeutics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan
| | - Taisuke Ishikawa
- Omics Research Center, National Cerebral and Cardiovascular Center, Suita, Japan
| | - Naomasa Makita
- Omics Research Center, National Cerebral and Cardiovascular Center, Suita, Japan
| | - Masahito Hitosugi
- Department of Legal Medicine, Shiga University of Medical Science, Otsu, Japan
| | - Hiroshi Matsuura
- Division of Cell Physiology, Department of Physiology, Shiga University of Medical Science, Otsu, Japan
| | - Seiko Ohno
- Department of Bioscience and Genetics, National Cerebral and Cardiovascular Center, Suita, Japan.,Center for Epidemiologic Research in Asia, Shiga University of Medical Science, Otsu, Japan
| | - Minoru Horie
- Center for Epidemiologic Research in Asia, Shiga University of Medical Science, Otsu, Japan
| | - Hisakazu Ogita
- Division of Molecular Medical Biochemistry, Department of Biochemistry and Molecular Biology, Shiga University of Medical Science, Otsu, Japan
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24
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Lu S, Liao Z, Lu X, Katschinski DM, Mercola M, Chen J, Heller Brown J, Molkentin JD, Bossuyt J, Bers DM. Hyperglycemia Acutely Increases Cytosolic Reactive Oxygen Species via O-linked GlcNAcylation and CaMKII Activation in Mouse Ventricular Myocytes. Circ Res 2020; 126:e80-e96. [PMID: 32134364 DOI: 10.1161/circresaha.119.316288] [Citation(s) in RCA: 101] [Impact Index Per Article: 20.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
RATIONALE Diabetes mellitus is a complex, multisystem disease, affecting large populations worldwide. Chronic CaMKII (Ca2+/calmodulin-dependent kinase II) activation may occur in diabetes mellitus and be arrhythmogenic. Diabetic hyperglycemia was shown to activate CaMKII by (1) O-linked attachment of N-acetylglucosamine (O-GlcNAc) at S280 leading to arrhythmia and (2) a reactive oxygen species (ROS)-mediated oxidation of CaMKII that can increase postinfarction mortality. OBJECTIVE To test whether high extracellular glucose (Hi-Glu) promotes ventricular myocyte ROS generation and the role played by CaMKII. METHODS AND RESULTS We tested how extracellular Hi-Glu influences ROS production in adult ventricular myocytes, using DCF (2',7'-dichlorodihydrofluorescein diacetate) and genetically targeted Grx-roGFP2 redox sensors. Hi-Glu (30 mmol/L) significantly increased the rate of ROS generation-an effect prevented in myocytes pretreated with CaMKII inhibitor KN-93 or from either global or cardiac-specific CaMKIIδ KO (knockout) mice. CaMKII KO or inhibition also prevented Hi-Glu-induced sarcoplasmic reticulum Ca2+ release events (Ca2+ sparks). Thus, CaMKII activation is required for Hi-Glu-induced ROS generation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes. To test the involvement of O-GlcNAc-CaMKII pathway, we inhibited GlcNAcylation removal by Thiamet G (ThmG), which mimicked the Hi-Glu-induced ROS production. Conversely, inhibition of GlcNAcylation (OSMI-1 [(αR)-α-[[(1,2-dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmethyl)-benzeneacetamide]) prevented ROS induction in response to either Hi-Glu or ThmG. Moreover, in a CRSPR-based knock-in mouse in which the functional GlcNAcylation site on CaMKIIδ was ablated (S280A), neither Hi-Glu nor ThmG induced myocyte ROS generation. So CaMKIIδ-S280 is required for the Hi-Glu-induced (and GlcNAc dependent) ROS production. To identify the ROS source(s), we used different inhibitors of NOX (NADPH oxidase) 2 (Gp91ds-tat peptide), NOX4 (GKT137831), mitochondrial ROS (MitoTempo), and NOS (NO synthase) pathway inhibitors (L-NAME, L-NIO, and L-NPA). Only NOX2 inhibition or KO prevented Hi-Glu/ThmG-induced ROS generation. CONCLUSIONS Diabetic hyperglycemia induces acute cardiac myocyte ROS production by NOX2 that requires O-GlcNAcylation of CaMKIIδ at S280. This novel ROS induction may exacerbate pathological consequences of diabetic hyperglycemia.
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Affiliation(s)
- Shan Lu
- From the Department of Pharmacology, University of California, Davis School of Medicine (S.L., Z.L., J.B., D.M.B.)
| | - Zhandi Liao
- From the Department of Pharmacology, University of California, Davis School of Medicine (S.L., Z.L., J.B., D.M.B.)
| | - Xiyuan Lu
- Department of Cardiology, Renji Hospital School of Medicine, Jiaotong University, Shanghai, China (X.L.)
| | - Dörthe M Katschinski
- Institute of Cardiovascular Physiology, University Medical Centre Göttingen, Germany (D.M.K.)
- German Center for Cardiovascular Research, Partner Site, Göttingen (D.M.K.)
| | - Mark Mercola
- Stanford Cardiovascular Institute and Department of Medicine, Stanford University, CA (M.M.)
| | - Ju Chen
- Department of Medicine (J.C.), University of California San Diego, La Jolla
| | - Joan Heller Brown
- Department of Pharmacology (J.H.B.), University of California San Diego, La Jolla
| | - Jeffery D Molkentin
- Department of Pediatrics, University of Cincinnati, Cincinnati Children's Hospital Medical Center, OH (J.D.M.)
| | - Julie Bossuyt
- From the Department of Pharmacology, University of California, Davis School of Medicine (S.L., Z.L., J.B., D.M.B.)
| | - Donald M Bers
- From the Department of Pharmacology, University of California, Davis School of Medicine (S.L., Z.L., J.B., D.M.B.)
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25
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Abstract
Cre-mediated recombination has become a powerful tool to confine gene deletions (conditional knockouts) or overexpression of genes (conditional knockin/overexpression). By spatiotemporal restriction of genetic manipulations, major problems of classical knockouts such as embryonic lethality or pleiotropy can be circumvented. Furthermore, Cre-mediated recombination has broad applications in the analysis of the cellular behavior of subpopulations and cell types as well as for genetic fate mapping. This chapter gives an overview about applications for the Cre/LoxP system and their execution.
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Affiliation(s)
- Claudius F Kratochwil
- Zoology and Evolutionary Biology, Department of Biology, University of Konstanz, Konstanz, Germany
| | - Filippo M Rijli
- Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
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26
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Prykhozhij SV, Fuller C, Steele SL, Veinotte CJ, Razaghi B, Robitaille JM, McMaster CR, Shlien A, Malkin D, Berman JN. Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9. Nucleic Acids Res 2019; 46:e102. [PMID: 29905858 PMCID: PMC6158492 DOI: 10.1093/nar/gky512] [Citation(s) in RCA: 40] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2018] [Accepted: 05/23/2018] [Indexed: 12/21/2022] Open
Abstract
We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.
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Affiliation(s)
- Sergey V Prykhozhij
- Departments of Pediatrics, Microbiology & Immunology, and Pathology, Dalhousie University, Halifax, NS, B3H 4R2, Canada
| | - Charlotte Fuller
- Michael G. DeGroote School of Medicine, McMaster University,Hamilton, ON, L8S4L8, Canada
| | | | - Chansey J Veinotte
- Departments of Pediatrics, Microbiology & Immunology, and Pathology, Dalhousie University, Halifax, NS, B3H 4R2, Canada
| | - Babak Razaghi
- Departments of Pediatrics, Microbiology & Immunology, and Pathology, Dalhousie University, Halifax, NS, B3H 4R2, Canada
| | - Johane M Robitaille
- Departments of Pediatrics, Microbiology & Immunology, and Pathology, Dalhousie University, Halifax, NS, B3H 4R2, Canada
| | - Christopher R McMaster
- Departments of Pediatrics, Microbiology & Immunology, and Pathology, Dalhousie University, Halifax, NS, B3H 4R2, Canada
| | - Adam Shlien
- Departments of Pediatrics and Medical Biophysics, University of Toronto, Toronto, ON, M5G 1X8, Canada
| | - David Malkin
- Departments of Pediatrics and Medical Biophysics, University of Toronto, Toronto, ON, M5G 1X8, Canada
| | - Jason N Berman
- Departments of Pediatrics, Microbiology & Immunology, and Pathology, Dalhousie University, Halifax, NS, B3H 4R2, Canada
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27
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Zarei A, Razban V, Hosseini SE, Tabei SMB. Creating cell and animal models of human disease by genome editing using CRISPR/Cas9. J Gene Med 2019; 21:e3082. [PMID: 30786106 DOI: 10.1002/jgm.3082] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2018] [Revised: 02/02/2019] [Accepted: 02/02/2019] [Indexed: 12/26/2022] Open
Affiliation(s)
- Ali Zarei
- Department of Molecular Genetics, Marvdasht BranchIslamic Azad University Marvdasht Iran
- Department of Molecular Genetics, Science and Research BranchIslamic Azad University Fars Iran
| | - Vahid Razban
- Department of Molecular medicine, School of Advanced Medical Sciences and Technologies Shiraz Iran
- Stem Cell and Transgenic Technology Research CenterShiraz University of Medical Sciences Shiraz Iran
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28
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Homanics GE. Gene-edited CRISPy Critters for alcohol research. Alcohol 2019; 74:11-19. [PMID: 30621855 PMCID: PMC6334660 DOI: 10.1016/j.alcohol.2018.03.001] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2018] [Revised: 03/01/2018] [Accepted: 03/02/2018] [Indexed: 12/26/2022]
Abstract
Genetically engineered animals are powerful tools that have provided invaluable insights into mechanisms of alcohol action and alcohol-use disorder. Traditionally, production of gene-targeted animals was a tremendously expensive, time consuming, and technically demanding undertaking. However, the recent advent of facile methods for editing the genome at very high efficiency is revolutionizing how these animals are made. While pioneering approaches to create gene-edited animals first used zinc finger nucleases and subsequently used transcription activator-like effector nucleases, these approaches have been largely supplanted in an extremely short period of time with the recent discovery and precocious maturation of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. CRISPR uses a short RNA sequence to guide a non-specific CRISPR-associated nuclease (Cas) to a precise, single location in the genome. Because the CRISPR/Cas system can be cheaply, rapidly, and easily reprogrammed to target nearly any genomic locus of interest simply by recoding the sequence of the guide RNA, this gene-editing system has been rapidly adopted by numerous labs around the world. With CRISPR/Cas, it is now possible to perform gene editing directly in early embryos from every species of animals that is of interest to the alcohol field. Techniques have been developed that enable the rapid production of animals in which a gene has been inactivated (knockout) or modified to harbor specific nucleotide changes (knockins). This system has also been used to insert specific DNA sequences such as reporter or recombinase genes into specific loci of interest. Genetically engineered animals created with the CRISPR/Cas system (CRISPy Critters) are being produced at an astounding pace. Animal production is no longer a significant bottleneck to new discoveries. CRISPy animal studies are just beginning to appear in the alcohol literature, but their use is expected to explode in the near future. CRISPy mice, rats, and other model organisms are sure to facilitate advances in our understanding of alcohol-use disorder.
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Affiliation(s)
- Gregg E Homanics
- Department of Anesthesiology, University of Pittsburgh School of Medicine, 6060 Biomedical Science Tower-3, 3501 Fifth Avenue, Pittsburgh, PA 15261, United States; Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, 6060 Biomedical Science Tower-3, 3501 Fifth Avenue, Pittsburgh, PA 15261, United States; Department of Neurobiology, University of Pittsburgh School of Medicine, 6060 Biomedical Science Tower-3, 3501 Fifth Avenue, Pittsburgh, PA 15261, United States.
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29
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Yamamoto Y, Gerbi SA. Making ends meet: targeted integration of DNA fragments by genome editing. Chromosoma 2018; 127:405-420. [PMID: 30003320 PMCID: PMC6330168 DOI: 10.1007/s00412-018-0677-6] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2018] [Revised: 06/25/2018] [Accepted: 06/28/2018] [Indexed: 12/27/2022]
Abstract
Targeted insertion of large pieces of DNA is an important goal of genetic engineering. However, this goal has been elusive since classical methods for homology-directed repair are inefficient and often not feasible in many systems. Recent advances are described here that enable site-specific genomic insertion of relatively large DNA with much improved efficiency. Using the preferred repair pathway in the cell of nonhomologous end-joining, DNA of up to several kb could be introduced with remarkably good precision by the methods of HITI and ObLiGaRe with an efficiency up to 30-40%. Recent advances utilizing homology-directed repair (methods of PITCh; short homology arms including ssODN; 2H2OP) have significantly increased the efficiency for DNA insertion, often to 40-50% or even more depending on the method and length of DNA. The remaining challenges of integration precision and off-target site insertions are summarized. Overall, current advances provide major steps forward for site-specific insertion of large DNA into genomes from a broad range of cells and organisms.
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Affiliation(s)
- Yutaka Yamamoto
- Department of Molecular Biology, Cell Biology and Biochemistry, Brown University Division of Biology and Medicine, Sidney Frank Hall room 260, 185 Meeting Street, Providence, RI, 02912, USA
| | - Susan A Gerbi
- Department of Molecular Biology, Cell Biology and Biochemistry, Brown University Division of Biology and Medicine, Sidney Frank Hall room 260, 185 Meeting Street, Providence, RI, 02912, USA.
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30
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Fang L, Hung SSC, Yek J, El Wazan L, Nguyen T, Khan S, Lim SY, Hewitt AW, Wong RCB. A Simple Cloning-free Method to Efficiently Induce Gene Expression Using CRISPR/Cas9. MOLECULAR THERAPY-NUCLEIC ACIDS 2018; 14:184-191. [PMID: 30594894 PMCID: PMC6307107 DOI: 10.1016/j.omtn.2018.11.008] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/20/2018] [Revised: 11/15/2018] [Accepted: 11/15/2018] [Indexed: 12/13/2022]
Abstract
Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here, we report a simple, cloning-free method to induce gene expression at an endogenous locus using CRISPR/Cas9 activators. Our strategy utilizes synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65, and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1, and NEUROD1) in human cells, whereas the rat cells exhibit more variable and less-efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applied as a rapid workflow to initiate gain-of-function studies for a range of molecular- and cell-biology disciplines.
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Affiliation(s)
- Lyujie Fang
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia; Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, VIC, Australia; Jinan University, Guangzhou, China
| | - Sandy S C Hung
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia
| | - Jennifer Yek
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia; Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, VIC, Australia
| | - Layal El Wazan
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia; Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, VIC, Australia
| | - Tu Nguyen
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia; Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, VIC, Australia
| | - Shahnaz Khan
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia
| | - Shiang Y Lim
- O'Brien Institute Department, St Vincent's Institute of Medical Research, Fitzroy, VIC, Australia
| | - Alex W Hewitt
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia; Menzies Institute for Medical Research, School of Medicine, University of Tasmania, Hobart, TAS, Australia
| | - Raymond C B Wong
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia; Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, VIC, Australia; Shenzhen Eye Hospital, School of Medicine, Shenzhen University, Shenzhen, China.
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31
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Burg L, Palmer N, Kikhi K, Miroshnik ES, Rueckert H, Gaddy E, MacPherson Cunningham C, Mattonet K, Lai SL, Marín-Juez R, Waring RB, Stainier DYR, Balciunas D. Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish. PLoS Genet 2018; 14:e1007754. [PMID: 30427827 PMCID: PMC6261631 DOI: 10.1371/journal.pgen.1007754] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2018] [Revised: 11/28/2018] [Accepted: 10/10/2018] [Indexed: 02/07/2023] Open
Abstract
Many eukaryotic genes play essential roles in multiple biological processes in several different tissues. Conditional mutants are needed to analyze genes with such pleiotropic functions. In vertebrates, conditional gene inactivation has only been feasible in the mouse, leaving other model systems to rely on surrogate experimental approaches such as overexpression of dominant negative proteins and antisense-based tools. Here, we have developed a simple and straightforward method to integrate loxP sequences at specific sites in the zebrafish genome using the CRISPR/Cas9 technology and oligonucleotide templates for homology directed repair. We engineered conditional (floxed) mutants of tbx20 and fleer, and demonstrate excision of exons flanked by loxP sites using tamoxifen-inducible CreERT2 recombinase. To demonstrate broad applicability of our method, we also integrated loxP sites into two additional genes, aldh1a2 and tcf21. The ease of this approach will further expand the use of zebrafish to study various aspects of vertebrate biology, especially post-embryonic processes such as regeneration. Some genes are expressed and function in a single tissue, and the effect of their loss on that tissue can be readily determined. Frequently, however, genes that are necessary for the development or maintenance of one tissue are also important for other tissues or cell types. Genes of the latter type are difficult to analyze because of the complications resulting from an organism having multiple defects in different tissues. The solution pioneered by mouse geneticists is to inactivate the gene of interest in only one tissue at a time. This elegant approach requires the ability to make specific edits to the genome, a technology that was not readily available to zebrafish researchers until recently. Using the CRISPR/Cas9 genome editing tool, we have developed a simple, reliable, and efficient method to insert DNA sequences into the zebrafish genome that enable conditional gene inactivation. Our methodology will be useful not only for the study of genes that play important roles in multiple tissues, but also for the genetic analysis of biological processes which occur in adult animals.
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Affiliation(s)
- Leonard Burg
- Department of Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America
| | - Nicholas Palmer
- Department of Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America
| | - Khrievono Kikhi
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
| | - Evgeniya S. Miroshnik
- Department of Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America
| | - Helen Rueckert
- Department of Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America
| | - Eleanor Gaddy
- Department of Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America
| | - Carlee MacPherson Cunningham
- Department of Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America
| | - Kenny Mattonet
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
| | - Shih-Lei Lai
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
| | - Rubén Marín-Juez
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
| | - Richard B. Waring
- Department of Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America
| | - Didier Y. R. Stainier
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
| | - Darius Balciunas
- Department of Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America
- * E-mail:
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32
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Prykhozhij SV, Fuller C, Steele SL, Veinotte CJ, Razaghi B, Robitaille JM, McMaster CR, Shlien A, Malkin D, Berman JN. Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9. Nucleic Acids Res 2018; 46:e102. [PMID: 29905858 PMCID: PMC6158492 DOI: 10.1093/nar/gky512 10.1093/nar/gky674] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2018] [Revised: 03/28/2018] [Accepted: 05/23/2018] [Indexed: 01/19/2024] Open
Abstract
We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.
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Affiliation(s)
- Sergey V Prykhozhij
- Departments of Pediatrics, Microbiology & Immunology, and Pathology, Dalhousie University, Halifax, NS, B3H 4R2, Canada
| | - Charlotte Fuller
- Michael G. DeGroote School of Medicine, McMaster University,Hamilton, ON, L8S4L8, Canada
| | | | - Chansey J Veinotte
- Departments of Pediatrics, Microbiology & Immunology, and Pathology, Dalhousie University, Halifax, NS, B3H 4R2, Canada
| | - Babak Razaghi
- Departments of Pediatrics, Microbiology & Immunology, and Pathology, Dalhousie University, Halifax, NS, B3H 4R2, Canada
| | - Johane M Robitaille
- Departments of Pediatrics, Microbiology & Immunology, and Pathology, Dalhousie University, Halifax, NS, B3H 4R2, Canada
| | - Christopher R McMaster
- Departments of Pediatrics, Microbiology & Immunology, and Pathology, Dalhousie University, Halifax, NS, B3H 4R2, Canada
| | - Adam Shlien
- Departments of Pediatrics and Medical Biophysics, University of Toronto, Toronto, ON, M5G 1X8, Canada
| | - David Malkin
- Departments of Pediatrics and Medical Biophysics, University of Toronto, Toronto, ON, M5G 1X8, Canada
| | - Jason N Berman
- Departments of Pediatrics, Microbiology & Immunology, and Pathology, Dalhousie University, Halifax, NS, B3H 4R2, Canada
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33
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Lanza DG, Gaspero A, Lorenzo I, Liao L, Zheng P, Wang Y, Deng Y, Cheng C, Zhang C, Seavitt JR, DeMayo FJ, Xu J, Dickinson ME, Beaudet AL, Heaney JD. Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles. BMC Biol 2018; 16:69. [PMID: 29925370 PMCID: PMC6011517 DOI: 10.1186/s12915-018-0529-0] [Citation(s) in RCA: 59] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2017] [Accepted: 05/09/2018] [Indexed: 01/29/2023] Open
Abstract
BACKGROUND The International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene-phenotype associations. While CRISPR/Cas9-mediated null allele production in mice is highly efficient, generation of conditional alleles has proven to be more difficult. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-initiated homology-driven repair (HDR) with short and long single stranded oligodeoxynucleotides (ssODNs and lssDNAs). RESULTS Using pairs of single guide RNAs and short ssODNs to introduce loxP sites around a critical exon or exons, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 out of 30 targeted genes. LoxP sites integrated in cis in at least one mouse for 18 of 23 genes. However, loxP sites were mutagenized in 4 of the 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was minimally influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and single lssDNAs to introduce loxP-flanked exons, conditional allele founders were generated for all four genes targeted, although one founder was found to harbor undesired mutations within the lssDNA sequence interval. Importantly, when employing either ssODNs or lssDNAs, random integration events were detected. CONCLUSIONS Our studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssDNAs are amenable to high-throughput production of conditional alleles when they can be employed. Regardless of the single-stranded donor utilized, it is essential to screen for sequence errors at sites of HDR and random insertion of donor sequences into the genome.
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Affiliation(s)
- Denise G Lanza
- Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, MS BCM225, Houston, TX, 77030, USA
- Mouse ES Cell Core, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA
| | - Angelina Gaspero
- Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, MS BCM225, Houston, TX, 77030, USA
| | - Isabel Lorenzo
- Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, MS BCM225, Houston, TX, 77030, USA
- Mouse ES Cell Core, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA
| | - Lan Liao
- Department of Molecular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA
- Genetically Engineered Mouse Core, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA
| | - Ping Zheng
- Mouse ES Cell Core, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA
| | - Ying Wang
- Mouse ES Cell Core, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA
| | - Yu Deng
- Lester and Sue Smith Breast Center, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA
| | - Chonghui Cheng
- Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, MS BCM225, Houston, TX, 77030, USA
- Department of Molecular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA
- Lester and Sue Smith Breast Center, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA
| | - Chuansheng Zhang
- Department of Neuroscience, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA
| | - John R Seavitt
- Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, MS BCM225, Houston, TX, 77030, USA
| | - Francesco J DeMayo
- Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, Durham, NC, 27709, USA
| | - Jianming Xu
- Department of Molecular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA
- Genetically Engineered Mouse Core, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA
| | - Mary E Dickinson
- Department of Molecular Physiology and Biophysics, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA
| | - Arthur L Beaudet
- Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, MS BCM225, Houston, TX, 77030, USA
| | - Jason D Heaney
- Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, MS BCM225, Houston, TX, 77030, USA.
- Mouse ES Cell Core, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA.
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34
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Stroud MJ, Fang X, Zhang J, Guimarães-Camboa N, Veevers J, Dalton ND, Gu Y, Bradford WH, Peterson KL, Evans SM, Gerace L, Chen J. Luma is not essential for murine cardiac development and function. Cardiovasc Res 2018; 114:378-388. [PMID: 29040414 PMCID: PMC6019056 DOI: 10.1093/cvr/cvx205] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/19/2017] [Accepted: 10/11/2017] [Indexed: 12/13/2022] Open
Abstract
AIMS Luma is a recently discovered, evolutionarily conserved protein expressed in mammalian heart, which is associated with the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex. The LINC complex structurally integrates the nucleus and the cytoplasm and plays a critical role in mechanotransduction across the nuclear envelope. Mutations in several LINC components in both humans and mice result in various cardiomyopathies, implying they play essential, non-redundant roles. A single amino acid substitution of serine 358 to leucine (S358L) in Luma is the unequivocal cause of a distinct form of arrhythmogenic cardiomyopathy. However, the role of Luma in heart has remained obscure. In addition, it also remains to be determined how the S358L mutation in Luma leads to cardiomyopathy. METHODS AND RESULTS To determine the role of Luma in the heart, we first determined the expression pattern of Luma in mouse heart. Luma was sporadically expressed in cardiomyocytes throughout the heart, but was highly and uniformly expressed in cardiac fibroblasts and vascular smooth muscle cells. We also generated germline null Luma mice and discovered that germline null mutants were viable and exhibited normal cardiac function. Luma null mice also responded normally to pressure overload induced by transverse aortic constriction. In addition, localization and expression of other LINC complex components in both cardiac myocytes and fibroblasts was unaffected by global loss of Luma. Furthermore, we also generated and characterized Luma S358L knock-in mice, which displayed normal cardiac function and morphology. CONCLUSION Our data suggest that Luma is dispensable for murine cardiac development and function and that the Luma S358L mutation alone may not cause cardiomyopathy in mice.
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MESH Headings
- Animals
- Arrhythmogenic Right Ventricular Dysplasia/genetics
- Arrhythmogenic Right Ventricular Dysplasia/metabolism
- Cells, Cultured
- Cytoskeleton/metabolism
- Female
- Fibroblasts/metabolism
- Gene Expression Regulation, Developmental
- Genetic Predisposition to Disease
- Heart/embryology
- Heart/physiopathology
- Humans
- Hypertrophy, Left Ventricular/genetics
- Hypertrophy, Left Ventricular/metabolism
- Hypertrophy, Left Ventricular/physiopathology
- Male
- Mechanotransduction, Cellular
- Membrane Proteins/genetics
- Membrane Proteins/metabolism
- Mice, Inbred C57BL
- Mice, Knockout
- Morphogenesis
- Myocardium/metabolism
- Myocardium/pathology
- Myocytes, Cardiac/metabolism
- Myocytes, Smooth Muscle/metabolism
- Nuclear Matrix/metabolism
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Affiliation(s)
- Matthew J Stroud
- Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
| | - Xi Fang
- Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
| | - Jianlin Zhang
- Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
| | - Nuno Guimarães-Camboa
- Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093, USA
| | - Jennifer Veevers
- Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
| | - Nancy D Dalton
- Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
| | - Yusu Gu
- Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
| | - William H Bradford
- Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
| | - Kirk L Peterson
- Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
| | - Sylvia M Evans
- Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093, USA
| | - Larry Gerace
- Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Ju Chen
- Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
- Corresponding author. Tel: 858 822 4276; fax: 858 822 3027, E-mail:
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35
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Stroud MJ, Fang X, Veevers J, Chen J. Generation and Analysis of Striated Muscle Selective LINC Complex Protein Mutant Mice. Methods Mol Biol 2018; 1840:251-281. [PMID: 30141050 PMCID: PMC6887482 DOI: 10.1007/978-1-4939-8691-0_18] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/23/2023]
Abstract
The linker of nucleoskeleton and cytoskeleton (LINC) complex mediates intracellular cross talk between the nucleus and the cytoplasm. In striated muscle, the LINC complex provides structural support to the myocyte nucleus and plays an essential role in regulating gene expression and mechanotransduction. A wide range of cardiac and skeletal myopathies have been linked to mutations in LINC complex proteins. Studies utilizing tissue-specific knockout and mutant mouse models have revealed important insights into the roles of the LINC complex in striated muscle. In this chapter, we describe several feasible approaches for generating striated muscle-specific gene knockout and mutant mouse models to study LINC complex protein function in cardiac and skeletal muscle. The experimental procedures used for phenotyping and analysis of LINC complex knockout mice are also described.
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Affiliation(s)
- Matthew J Stroud
- Department of Medicine, University of California, San Diego, La Jolla, CA, USA
- Cardiovascular Division, King's College London, British Heart Foundation Centre of Excellence, London, UK
| | - Xi Fang
- Department of Medicine, University of California, San Diego, La Jolla, CA, USA
| | - Jennifer Veevers
- Department of Medicine, University of California, San Diego, La Jolla, CA, USA
| | - Ju Chen
- Department of Medicine, University of California, San Diego, La Jolla, CA, USA.
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36
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Luo Y, Li R, Wang J, Zhang M, Zou L, Ling L. An Ag+-stabilized triplex DNA molecular switch controlled hybridization chain reaction. Sci China Chem 2017. [DOI: 10.1007/s11426-017-9124-0] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
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37
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Horii T, Morita S, Kimura M, Terawaki N, Shibutani M, Hatada I. Efficient generation of conditional knockout mice via sequential introduction of lox sites. Sci Rep 2017; 7:7891. [PMID: 28801621 PMCID: PMC5554182 DOI: 10.1038/s41598-017-08496-8] [Citation(s) in RCA: 51] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2017] [Accepted: 07/12/2017] [Indexed: 01/17/2023] Open
Abstract
Conditional knockout using Cre/lox is essential for functional analysis of genes. CRISPR/Cas in combination with two sets of guide RNAs and a single-stranded oligonucleotide enables simultaneous insertion of two lox sequences. However, this method induces double-strand breaks at two sites on the same chromosome, which causes an undesirable chromosomal deletion and reduces the flanked lox (flox) rate. To solve this problem, we investigated a method that sequentially introduces each lox sequence at the 1-cell and 2-cell embryonic stages, respectively. The sequential method was applied to both microinjection and electroporation systems. Sequential electroporation improved the flox efficiency compared with ordinary simultaneous microinjection, leading to a high yield of offspring with floxed alleles. Finally, we directly produced Cre/lox mice containing both the Cre transgene and floxed allele via sequential electroporation using Cre zygotes, which accelerated the generation of conditional knockout mice compared with the ordinary method.
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Affiliation(s)
- Takuro Horii
- Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8512, Japan
| | - Sumiyo Morita
- Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8512, Japan
| | - Mika Kimura
- Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8512, Japan
| | - Naomi Terawaki
- Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8512, Japan
| | - Mihiro Shibutani
- Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8512, Japan
| | - Izuho Hatada
- Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8512, Japan.
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38
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CRISPR/Cas9-Mediated Deletion of Foxn1 in NOD/SCID/IL2rg -/- Mice Results in Severe Immunodeficiency. Sci Rep 2017; 7:7720. [PMID: 28798321 PMCID: PMC5552779 DOI: 10.1038/s41598-017-08337-8] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2017] [Accepted: 07/11/2017] [Indexed: 12/17/2022] Open
Abstract
Immunodeficient mice engrafted with either normal or cancerous human cells are widely used in basic and translational research. In particular, NOD/SCID/IL2rg−/− mice can support the growth of various types of human cancer cells. However, the hairs of these mice interfere with the observation and imaging of engrafted tissues. Therefore, novel hairless strains exhibiting comparable immunodeficiency would be beneficial. Recently, the CRISPR/Cas9 system has been used for efficient multiplexed genome editing. In the present study, we generated a novel strain of nude NOD/SCID/IL2rg−/− (NSIN) mice by knocking out Foxn1 from NOD/SCID/IL2rg−/− (NSI) mice using the CRISPR/Cas9 system. The NSIN mice were deficient in B, T, and NK cells and not only showed impaired T cell reconstitution and thymus regeneration after allogeneic bone marrow nucleated cell transplantation but also exhibited improved capacity to graft both leukemic and solid tumor cells compared with NSI, NOG, and NDG mice. Moreover, the NSIN mice facilitated the monitoring and in vivo imaging of both leukemia and solid tumors. Therefore, our NSIN mice provide a new platform for xenograft mouse models in basic and translational research.
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39
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Steward CA, Parker APJ, Minassian BA, Sisodiya SM, Frankish A, Harrow J. Genome annotation for clinical genomic diagnostics: strengths and weaknesses. Genome Med 2017; 9:49. [PMID: 28558813 PMCID: PMC5448149 DOI: 10.1186/s13073-017-0441-1] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
The Human Genome Project and advances in DNA sequencing technologies have revolutionized the identification of genetic disorders through the use of clinical exome sequencing. However, in a considerable number of patients, the genetic basis remains unclear. As clinicians begin to consider whole-genome sequencing, an understanding of the processes and tools involved and the factors to consider in the annotation of the structure and function of genomic elements that might influence variant identification is crucial. Here, we discuss and illustrate the strengths and weaknesses of approaches for the annotation and classification of important elements of protein-coding genes, other genomic elements such as pseudogenes and the non-coding genome, comparative-genomic approaches for inferring gene function, and new technologies for aiding genome annotation, as a practical guide for clinicians when considering pathogenic sequence variation. Complete and accurate annotation of structure and function of genome features has the potential to reduce both false-negative (from missing annotation) and false-positive (from incorrect annotation) errors in causal variant identification in exome and genome sequences. Re-analysis of unsolved cases will be necessary as newer technology improves genome annotation, potentially improving the rate of diagnosis.
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Affiliation(s)
- Charles A Steward
- Congenica Ltd, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1DR, UK. .,The Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
| | | | - Berge A Minassian
- Department of Pediatrics (Neurology), University of Texas Southwestern, Dallas, TX, USA.,Program in Genetics and Genome Biology and Department of Paediatrics (Neurology), The Hospital for Sick Children and University of Toronto, Toronto, Canada
| | - Sanjay M Sisodiya
- Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, London, WC1N 3BG, UK.,Chalfont Centre for Epilepsy, Chesham Lane, Chalfont St Peter, Buckinghamshire, SL9 0RJ, UK
| | - Adam Frankish
- The Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.,European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SD, UK
| | - Jennifer Harrow
- The Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.,Illumina Inc, Great Chesterford, Essex, CB10 1XL, UK
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