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

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).