PUBLICATION

Multiple genome modifications by the CRISPR/Cas9 system in zebrafish

Authors
Ota, S., Hisano, Y., Ikawa, Y., Kawahara, A.
ID
ZDB-PUB-140523-8
Date
2014
Source
Genes to cells : devoted to molecular & cellular mechanisms   19(7): 555-64 (Journal)
Registered Authors
Kawahara, Atsuo, Ota, Satoshi
Keywords
none
MeSH Terms
  • Animals
  • CRISPR-Cas Systems*
  • Embryo, Nonmammalian
  • Genetic Engineering/methods
  • Genetic Loci
  • Genome*
  • Heart/embryology
  • INDEL Mutation*
  • Inverted Repeat Sequences
  • Phenotype
  • Pigmentation/genetics
  • Retinal Pigment Epithelium/embryology
  • Zebrafish/embryology
  • Zebrafish/genetics*
  • Zebrafish Proteins/genetics*
PubMed
24848337 Full text @ Genes Cells
Abstract
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 (gRNAs) and the nuclease Cas9 mRNA. 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.
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping