CRISPR-Cas9介导的斑马鱼心脏中的精确敲入编辑
Summary
该协议描述了一种使用CRISPR-Cas9技术促进斑马鱼胚胎精确敲入编辑的方法。提出了一个表型管道，以证明这些技术对长QT综合征相关基因变异进行建模的适用性。
Abstract
动物模型中成簇规则间隔的短回文重复序列（CRISPR）能够为生理现象的研究提供精确的遗传操作。斑马鱼已被用作有效的遗传模型，在全器官和生物体水平上研究与遗传性疾病，发育和毒理学相关的许多问题。由于斑马鱼基因组注释和映射良好，已经开发了许多用于基因编辑的工具。然而，使用CRISPR生成和检测精确敲入编辑的有效性是一个限制因素。这里描述的是一种基于CRISPR-Cas9的敲入方法，该方法可以简单地检测负责心脏复极化并与电障碍长QT综合征（LQTS）相关的基因的精确编辑。这种双单向导RNA（sgRNA）方法切除并替换了靶序列，并连接了基因编码的报告基因。通过描述野生型和基因编辑斑马鱼幼虫心脏电功能的非侵入性表型测量，证明了这种方法的实用性。这种方法能够有效地研究整个生物体中与疾病相关的变异。此外，该策略为插入选择的外源序列提供了可能性，例如报告基因、直系同源物或基因编辑器。
Introduction
动物模型中基于CRISPR的基因编辑策略能够在全生物体水平上研究遗传性疾病，发育和毒理学1，2，3。斑马鱼提供了一个强大的模型，在许多生理方面比小鼠或人类来源的细胞模型更接近人类4。斑马鱼已经使用了广泛的遗传工具和策略，用于正向5和反向遗传筛选6。斑马鱼的综合遗传图谱和注释促进了基因编辑方法，成为设计靶向基因敲除（KO）和精确敲入（KI）的主要技术7。
尽管如此，在斑马鱼中生成精确的KI编辑受到效率低和准确检测困难的限制。尽管转录因子样效应核酸酶（TALENs）已成功用于KIs 8并进行了优化，但CRISPR提供了一种改进的基因编辑策略，具有更简单的sgRNA靶向。许多研究已经使用CRISPR在斑马鱼9，10，11，12，13，14，15，16，17，18，19，20中生成精确的KI，尽管通过CRISPR介导的同源定向修复（HDR）产生的这些编辑往往效率低下，内在成功率低。需要基因分型作为主要筛查的比率9，10，14，21。这表明需要高效的斑马鱼KI CRISPR系统，以及用于检测精确编辑的可靠高通量系统。
本研究的目的是描述一个在斑马鱼心脏中产生精确心脏基因KI的平台，通过简单和高通量检测成功的编辑。描述了一种基于CRISPR-Cas9的双sgRNA外显子替代方法，该方法基于TALEN方法8。这种方法涉及使用双 sgRNA 向导切除靶序列，并用包含目标 KI 和遗传编码内含子报告基因的外源模板序列替换（图 1）。在靶基因内含子序列中整合基因编码的荧光报告基因可以有效地检测阳性编辑。然后描述了一个表型平台，用于评估斑马鱼幼虫的心脏电功能，以非侵入性地表征与遗传性LQTS相关的基因变异，遗传性LQTS是一种使个体易发生心脏性猝死的心脏电疾病。
这些方法将加强斑马鱼KI基因编辑的获取和使用，以模拟遗传疾病并解决生物学和生理学问题，例如绘制基因表达模式和发育调节。由于斑马鱼的心脏比小鼠模型更相似于人类心脏电生理特征，因此它们作为心脏病建模的遗传可处理系统可能特别有吸引力7，22，23。
Protocol
使用斑马鱼的研究是根据西蒙弗雷泽大学动物护理委员会和加拿大动物护理委员会的政策和程序进行的，并根据协议#1264K-18完成。 1. 用于精确编辑的CRISPR组件设计 为了设计将用于切除含有KI靶位点的序列的双sgRNA向导，首先鉴定目的基因的斑马鱼直系同源物。注意： 图2 概述了使用双sgRNA CRISPR-Cas9方法进行精确编辑的步骤。 <l…
Representative Results
这种双sgRNA外显子替代CRISPR方法的成功使用突出了对斑马鱼 zkcnh6a 基因中LQTS相关变体R56Q的精确编辑的引入和简单检测。 图6 显示了在单细胞胚胎阶段注射的具有代表性的3 dpf幼虫，如上所述的CRISPR组分。 图6A 显示了YFP mVenus报告基因在眼晶状体中的表达作为成功模板整合的阳性报告基因。图 6B，C 显示了分别从?…
Discussion
使用CRISPR-Cas9进行精确基因编辑的工程受到HDR机制及其高效检测效率低的挑战。本文描述了一种基于CRISPR-Cas9的双sgRNA外显子替换方法，该方法在斑马鱼中产生精确的编辑，并直接目视检测阳性编辑。这种方法的有效性通过在 zkcnh6a 基因中产生精确的编辑来证明。本文展示了如何使用心率、心包尺寸和心电图形态的非侵入性表型测量来评估基因编辑斑马鱼幼虫的心脏功能。这种方法，从引入?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了加拿大卫生研究院研究项目拨款（T.W.C.）和加拿大自然科学与工程研究理事会发现补助金（T.W.C.）的支持。
Materials
| Program | |||
| CRISPOR | TEFOR Infrastructure | ||
| ENSEMBL | European Bioinformatics Institute | ||
| ImageJ | National Institutes of Health (NIH) | ||
| Micro-Manager | Open Source (Github) | ||
| NEBiocalculator | New England Biolabs (NEB) | ||
| EQUIPMENT | |||
| 24-well Plate | VWR | ||
| 25 mm Petri Dish | VWR | ||
| Blackfly USB3 Camera | Teledyne FLIR | ||
| C1000 Thermal Cycler | Bio-Rad | ||
| Centrifuge 5415C | Eppendorf | ||
| EZNA Gel Extraction Kit | Omega Biotek | ||
| MAXIscript T7 Transcription Kit | Invitrogen | ||
| MaxQ 5000 Incubator | Barnstead Lab Line | ||
| Miniprep Kit | Qiagen | ||
| mMessage mMachine T7 Ultra Transcription Kit | Invitrogen | ||
| ND1000 Spectrophotometer | Nanodrop | ||
| PCR Purification Kit | Qiagen | ||
| PLI 100A Picoinjector | Harvard Apparatus | ||
| PowerPac Basic Power Supply | Bio-Rad | ||
| Stemi 305 Steroscope | Zeiss | ||
| Wide Mini Sub Cell GT Electrophoresis System | Bio-Rad | ||
| ZebTec Zebrafish Housing System | Tecniplast | ||
| SERVICES | |||
| Gene Synthesis | Genewiz | ||
| Sanger Sequencing | Genewiz | ||
| REAGENTS | |||
| 10β Competent Cells | NEB | ||
| 10X PCR Buffer | Qiagen | ||
| 100 mM Nucleotide Mixture | ABM | ||
| Ampicillin | Sigma | ||
| BamHI Endonuclease w/ buffer | NEB | ||
| BsaI Endonuclease w/ buffer | NEB | ||
| DR274 Plasmid (XL1 Blue bacterial agar stab) | Addgene | ||
| EcoRI Endonuclease w/ buffer | NEB | ||
| Glycerol | |||
| HEPES | Sigma | ||
| HindIII Endonuclease w/ buffer | NEB | ||
| Kanamycin | Sigma | ||
| Methylene Blue | Sigma | ||
| MLM3613 Plasmid (XL1 Blue bacterial agar stab) | Addgene | ||
| MS-222 (Tricaine) | Sigma | ||
| pKHR5 Plasmid (DH5α bacterial agar stab) | Addgene | ||
| PmeI Endonuclease w/ buffer | NEB | ||
| SalI Endonuclease w/ buffer | NEB | ||
| Sodium Hydroxide | Sigma | ||
| T4 Ligase w/ buffer | Sigma | ||
| Taq Polymerase | Qiagen | ||
| TE Buffer | Sigma | ||
| Tris Hydrochloride | Sigma | ||
| XhoI Endonuclease w/ buffer | NEB | ||
| RECIPES | |||
| Solution | Component | Supplier | |
| Annealing Buffer (pH 7.5-8.0) | 10 mM Tris | Sigma | |
| 50 mM NaCl | Sigma | ||
| 1 mM EDTA | Sigma | ||
| E3 Media (pH 7.2) | 5 mM NaCl | Sigma | |
| 0.17 mM KCl | Sigma | ||
| 0.33 mM CaCl2 | Sigma | ||
| 0.33 mM MgSO4 | Sigma | ||
| Injection Buffer (pH 7.5) | 20 mM HEPES | Sigma | |
| 150 mM KCl | Sigma |
References
- Zarei, A., Razban, V., Hosseini, S. E., Tabei, S. M. B. Creating cell and animal models of human disease by genome editing using CRISPR/Cas9. The Journal of Gene Medicine. 21 (4), 3082 (2019).
- Lee, H., Yoon, D. E., Kim, K. Genome editing methods in animal models. Animal Cells and Systems. 24 (1), 8-16 (2020).
- Li, Q., et al. Applications of genome editing technology in animal disease modeling and gene therapy. Computational and Structural Biotechnology Journal. 17, 689-698 (2019).
- Gut, P., Reischauer, S., Stainier, D. Y. R., Arnaout, R. Little fish, big data: Zebrafish as a model for cardiovascular and metabolic disease. Physiological Reviews. 97 (3), 889-938 (2017).
- Kegel, L., et al. Forward genetic screen using zebrafish to identify new genes involved in myelination. Oligodendrocytes: Methods and Protocols. 1936, 185-209 (2019).
- Shah, A. N., Davey, C. F., Whitebirch, A. C., Miller, A. C., Moens, C. B. Rapid reverse genetic screening using CRISPR in zebrafish. Nature Methods. 12 (6), 535-540 (2015).
- González-Rosa, J. M. Zebrafish models of cardiac disease: From fortuitous mutants to precision medicine. Circulation Research. 130 (12), 1803-1826 (2022).
- Hoshijima, K., Jurynec, M. J., Grunwald, D. J. Precise editing of the Zebrafish genome made simple and efficient. Developmental Cell. 36 (6), 654-667 (2016).
- Albadri, S., Del Bene, F., Revenu, C. Genome editing using CRISPR/Cas9-based knock-in approaches in zebrafish. Methods. 121-122, 77-85 (2017).
- Armstrong, G. A. B., et al. Homology directed knockin of point mutations in the zebrafish tardbp and fus genes in ALS using the CRISPR/Cas9 system. PLoS One. 11 (3), 0150188 (2016).
- Bai, H., et al. CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish. BMC Genomics. 21 (1), 67 (2020).
- de Vrieze, E., et al. Efficient generation of knock-in zebrafish models for inherited disorders using CRISPR-Cas9 ribonucleoprotein complexes. International Journal of Molecular Sciences. 22 (17), 9429 (2021).
- Eschstruth, A., Schneider-Maunoury, S., Giudicelli, F. Creation of zebrafish knock-in reporter lines in the nefma gene by Cas9-mediated homologous recombination. Genesis. 58 (1), 23340 (2020).
- Irion, U., Krauss, J., Nüsslein-Volhard, C. Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system. Development. 141 (24), 4827-4830 (2014).
- Kimura, Y., Hisano, Y., Kawahara, A., Higashijima, S. Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering. Scientific Reports. 4 (1), 6545 (2014).
- Levic, D. S., Yamaguchi, N., Wang, S., Knaut, H., Bagnat, M. Knock-in tagging in zebrafish facilitated by insertion into non-coding regions. Development. 148 (19), (2021).
- Prykhozhij, S. V., et al. Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9. Nucleic Acids Research. 46 (17), 102 (2018).
- Wierson, W. A., et al. Efficient targeted integration directed by short homology in zebrafish and mammalian cells. eLife. 9, 53968 (2020).
- Boel, A., et al. CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments. Disease Models & Mechanisms. 11 (10), (2018).
- Tessadori, F., et al. Effective CRISPR/Cas9-based nucleotide editing in zebrafish to model human genetic cardiovascular disorders. Disease Models & Mechanisms. 11 (10), (2018).
- Zhang, Y., Zhang, Z., Ge, W. An efficient platform for generating somatic point mutations with germline transmission in the zebrafish by CRISPR/Cas9-mediated gene editing. The Journal of Biological Chemistry. 293 (17), 6611-6622 (2018).
- Vornanen, M., Hassinen, M. Zebrafish heart as a model for human cardiac electrophysiology. Channels. 10 (2), 101-110 (2016).
- Nemtsas, P., Wettwer, E., Christ, T., Weidinger, G., Ravens, U. Adult zebrafish heart as a model for human heart? An electrophysiological study. Journal of Molecular and Cellular Cardiology. 48 (1), 161-171 (2010).
- Haeussler, M., et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biology. 17 (1), 148 (2016).
- Liang, X., Potter, J., Kumar, S., Ravinder, N., Chesnut, J. D. Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA. Journal of Biotechnology. 241, 136-146 (2017).
- Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L., Corn, J. E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nature Biotechnology. 34 (3), 339-344 (2016).
- Sambrook, J., Fritsch, E. F., Maniatis, T. Precipitation of Large RNAs with Lithium Chloride. Molecular Cloning: A Laboratory Manual, Book 3. E.15. , (1989).
- Sambrook, J., Fritsch, E. F., Maniatis, T. Agarose Gel Electrophoresis. Molecular Cloning: A Laboratory Manual, Book 1. , 3-20 (1989).
- Sorlien, E. L., Witucki, M. A., Ogas, J. Efficient production and identification of CRISPR/Cas9-generated gene knockouts in the model system Danio rerio. Journal of Visualized Experiments. (138), e56969 (2018).
- Avdesh, A., et al. Regular care and maintenance of a zebrafish (Danio rerio) laboratory: An introduction. Journal of Visualized Experiments. (69), e4196 (2012).
- Rosen, J. N., Sweeney, M. F., Mably, J. D. Microinjection of zebrafish embryos to analyze gene function. Journal of Visualized Experiments. (25), e1115 (2009).
- Kossack, M. E., Draper, B. W. Genetic regulation of sex determination and maintenance in zebrafish (Danio rerio). Current Topics in Developmental Biology. 134, 119-149 (2019).
- Tanaka, Y., et al. Functional analysis of KCNH2 gene mutations of type 2 long QT syndrome in larval zebrafish using microscopy and electrocardiography. Heart and Vessels. 34 (1), 159-166 (2019).
- Dhillon, S. S., et al. Optimisation of embryonic and larval ECG measurement in zebrafish for quantifying the effect of QT prolonging drugs. PLoS One. 8 (4), 60552 (2013).
- Yu, F., et al. Evolving cardiac conduction phenotypes in developing zebrafish larvae: Implications to drug sensitivity. Zebrafish. 7 (4), 325-331 (2010).
- Hurst, R. M. . Development and optimization of tools for embryonic electrocardiograph recording for heart dysfunction in zebrafish. , (2018).
- Meeker, N. D., Hutchinson, S. A., Ho, L., Trede, N. S. Method for isolation of PCR-ready genomic DNA from zebrafish tissues. BioTechniques. 43 (5), 610-614 (2007).
- Arnaout, R., et al. Zebrafish model for human long QT syndrome. Proceedings of the National Academy of Sciences of the United States of America. 104 (27), 11316-11321 (2007).
- Milan, D. J., Peterson, T. A., Ruskin, J. N., Peterson, R. T., MacRae, C. A. Drugs that induce repolarization abnormalities cause bradycardia in zebrafish. Circulation. 107 (10), 1355-1358 (2003).
- Langheinrich, U., Vacun, G., Wagner, T. Zebrafish embryos express an orthologue of HERG and are sensitive toward a range of QT-prolonging drugs inducing severe arrhythmia. Toxicology and Applied Pharmacology. 193 (3), 370-382 (2003).
- MacRae, C. A. Cardiac arrhythmia: In vivo screening in the zebrafish to overcome complexity in drug discovery. Expert Opinion on Drug Discovery. 5 (7), 619-632 (2010).
Cite This Article
Simpson, K. E., Faizi, S., Venkateshappa, R., Yip, M., Johal, R., Poburko, D., Cheng, Y. M., Hunter, D., Lin, E., Tibbits, G. F., Claydon, T. W. CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts. J. Vis. Exp. (187), e64209, doi:10.3791/64209 (2022).