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Use of CRISPR/Cas9 for the Modification of the Mouse Genome

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Target Identification and Validation in Drug Discovery

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1953))

Abstract

The use of CRISPR/Cas9 to modify the mouse genome has gained immense interest in the past few years since it allows the direct modification of embryos, bypassing the need of labor-intensive procedures for the manipulation of embryonic stem cells. By shortening the overall timelines and reducing the costs for the generation of new genetically modified mouse lines (Li et al., Nat Biotechnol 31: 681–683, 2013), this technology has rapidly become a major tool for in vivo drug discovery applications.

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References

  1. Gaj T, Gersbach CA, Barbas CF (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405

    Article  CAS  Google Scholar 

  2. Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821

    Article  CAS  Google Scholar 

  3. Ran FA, Hsu PD, Lin C-Y et al (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154:1380–1389

    Article  CAS  Google Scholar 

  4. Hsu PD, Scott DA, Weinstein JA et al (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832

    Article  CAS  Google Scholar 

  5. Miyaoka Y, Berman JR, Cooper SB et al (2016) Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Sci Rep 6:23549

    Article  CAS  Google Scholar 

  6. Wang H, Yang H, Shivalila CS et al (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918

    Article  CAS  Google Scholar 

  7. Zhang L, Jia R, Palange NJ et al (2015) Large genomic fragment deletions and insertions in mouse using CRISPR/Cas9. PLoS One 10:e0120396

    Article  Google Scholar 

  8. Sonoda E, Hochegger H, Saberi A et al (2006) Differential usage of non-homologous end-joining and homologous recombination in double strand break repair. DNA Repair 5:1021–1029

    Article  CAS  Google Scholar 

  9. Inui M, Miyado M, Igarashi M et al (2014) Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system. Sci Rep 4:5396

    Article  CAS  Google Scholar 

  10. Yang H, Wang H, Shivalila CS et al (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154:1370–1379

    Article  CAS  Google Scholar 

  11. Yoshimi K, Kunihiro Y, Kaneko T et al (2016) ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes. Nat Commun 7:10431

    Article  CAS  Google Scholar 

  12. Quadros RM, Miura H, Harms DW et al (2017) Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol 18:92

    Article  Google Scholar 

  13. Miura H, Gurumurthy CB, Sato T et al (2015) CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA. Sci Rep 5:12799

    Article  CAS  Google Scholar 

  14. Xiao A, Cheng Z, Kong L et al (2014) CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics 30:1180–1182

    Article  CAS  Google Scholar 

  15. Doench JG, Fusi N, Sullender M et al (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34:184–191

    Article  CAS  Google Scholar 

  16. Doench JG, Hartenian E, Graham DB et al (2014) Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol 32:1262–1267

    Article  CAS  Google Scholar 

  17. Haeussler M, Schoenig K, Eckert H et al (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17:148

    Article  Google Scholar 

  18. Hough SH, Kancleris K, Brody L et al (2017) Guide Picker is a comprehensive design tool for visualizing and selecting guides for CRISPR experiments. BMC Bioinf 18:167

    Article  Google Scholar 

  19. Montague TG, Cruz JM, Gagnon JA et al (2014) CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res 42:W401–W407

    Article  CAS  Google Scholar 

  20. Labun K, Montague TG, Gagnon JA et al (2016) CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res 44:W272–W276

    Article  CAS  Google Scholar 

  21. Sakurai T, Watanabe S, Kamiyoshi A et al (2014) A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice. BMC Biotechnol 14:69

    Article  Google Scholar 

  22. Fu Y, Foden JA, Khayter C et al (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31:822–826

    Article  CAS  Google Scholar 

  23. Yen S-T, Zhang M, Deng JM et al (2014) Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev Biol 393:3–9

    Article  CAS  Google Scholar 

  24. Li D, Qiu Z, Shao Y et al (2013) Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol 31:681–683

    Article  CAS  Google Scholar 

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Correspondence to Adriano Flora .

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Klimke, A., Güttler, S., Kuballa, P., Janzen, S., Ortmann, S., Flora, A. (2019). Use of CRISPR/Cas9 for the Modification of the Mouse Genome. In: Moll, J., Carotta, S. (eds) Target Identification and Validation in Drug Discovery. Methods in Molecular Biology, vol 1953. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9145-7_13

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  • DOI: https://doi.org/10.1007/978-1-4939-9145-7_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9144-0

  • Online ISBN: 978-1-4939-9145-7

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