Zebrafish pp 53-63 | Cite as

Detection of Multiple Genome Modifications Induced by the CRISPR/Cas9 System

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1451)

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).

Key words

Zebrafish CRISPR/Cas9 Genome modifications HMA Multi-locus HMA 

Notes

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).

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical ScienceUniversity of YamanashiChuoJapan

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