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Multiple genome modifications by the CRISPR /Cas9 system in zebrafish
Author(s) -
Ota Satoshi,
Hisano Yu,
Ikawa Yoshiya,
Kawahara Atsuo
Publication year - 2014
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/gtc.12154
Subject(s) - crispr , biology , cas9 , indel , genetics , trans activating crrna , gene , genome , locus (genetics) , zebrafish , indel mutation , genome editing , crispr interference , computational biology , single nucleotide polymorphism , genotype
The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated (Cas) system, which is an adaptive immune system of bacteria, has become a powerful tool for genome editing in various model organisms. Here, we demonstrate multiple genome modifications mediated by CRISPR/Cas9 in zebrafish ( Danio rerio ). Multiple genes including golden/gol and tyrosinase/tyr , which are involved in pigment formation, and s1pr2 and spns2 , which are involved in cardiac development, were disrupted with insertion and/or deletion (indel) mutations introduced by the co‐injection of multiple guide RNAs (g RNA s) and the nuclease Cas9 m RNA . We simultaneously observed two distinct phenotypes, such as, the two hearts phenotype and the hypopigmentation of skin melanophores and the retinal pigment epithelium, in the injected F0 embryos. Additionally, we detected the targeted deletion and inversion genes as a 7.1‐kb fragment between the two distinct spns2 targeted sites together with indel mutations. Conversely, chromosomal translocations among five target loci were not detected. Therefore, we confirmed that the CRISPR/Cas9‐induced indel mutations and a locus‐specific deletion were heritable in F1 embryos. To screen founders, we improved heteroduplex mobility assay (HMA) for simultaneously detecting indel mutations in different target loci. The results suggest that the multi‐locus HMA is a powerful tool for identification of multiple genome modifications mediated by the CRISPR/Cas9 system.