Abstract
The recent remarkable innovation of an RNA-guided nuclease system, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, enables us the modification of specific genomic loci in various model animals including zebrafish. With this system, multiple guide RNAs simultaneously injected with the Cas9 nuclease into zebrafish embryos cause multiple genome modifications at different genomic loci with high efficiency; therefore, a simple method to detect individual mutations at distinct loci is desired. In this chapter, we describe a procedure for inducing multiple CRISPR/Cas9-mediated genome modifications in zebrafish and a convenient method to detect CRISPR/Cas9-induced insertion and/or deletion (indel) mutations using a heteroduplex mobility assay (HMA).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482:331–338
Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826
**ek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821
Deveau H, Barrangou R, Garneau JE et al (2008) Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol 190:1390–1400
Mojica FJ, Diez-Villaseñor C, GarcÃa-MartÃnez J et al (2009) Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155:733–740
Hisano Y, Ota S, Kawahara A (2014) Genome editing using artificial site-specific nucleases in zebrafish. Dev Growth Differ 56:26–33
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
Jao LE, Wente SR, Chen W (2013) Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci U S A 110:13904–13909
Ota S, Hisano Y, Ikawa Y et al (2014) Multiple genome modifications by the CRISPR/Cas9 system in zebrafish. Genes Cells 19:555–564
Miller JC, Holmes MC, Wang J et al (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25:778–785
Kim HJ, Lee HJ, Kim H et al (2009) Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome Res 19:1279–1288
Dahlem TJ, Hoshijima K, Jurynec MJ et al (2012) Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome. PLoS Genet 8:e1002861
Ota S, Hisano Y, Muraki M et al (2013) Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays. Genes Cells 18:450–458
Hwang WY, Fu Y, Reyon D et al (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31:227–229
Ansai S, Kinoshita M (2014) Targeted mutagenesis using CRISPR/Cas system in medaka. Biol Open 3:362–371
Hwang WY, Fu Y, Reyon D et al (2013) Heritable and precise zebrafish genome editing using a CRISPR-Cas system. PLoS One 8:e68708
Acknowledgment
This work was supported by the Japan Society for the Promotion of Science and the Program for Next-Generation World-Leading Researchers (NEXT Program).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Ota, S., Kawahara, A. (2016). Detection of Multiple Genome Modifications Induced by the CRISPR/Cas9 System. In: Kawakami, K., Patton, E., Orger, M. (eds) Zebrafish. Methods in Molecular Biology, vol 1451. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3771-4_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3771-4_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3769-1
Online ISBN: 978-1-4939-3771-4
eBook Packages: Springer Protocols