Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

Li-En Jao, Susan R. Wente, Wenbiao Chen

Research output: Contribution to journalArticle

569 Citations (Scopus)

Abstract

A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Herewe report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.

Original languageEnglish (US)
Pages (from-to)13904-13909
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume110
Issue number34
DOIs
StatePublished - 2013
Externally publishedYes

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Clustered Regularly Interspaced Short Palindromic Repeats
Zebrafish
Mutagenesis
Mutation
CRISPR-Associated Proteins
Guide RNA
Phenotype
Gene Knockout Techniques
Germ-Line Mutation
Transgenes
Germ Cells
Codon
Fishes
Proteins
Genome
Gene Editing
Genes

Keywords

  • Genome engineering
  • Pigmentation
  • RNA-guided mutagenesis

ASJC Scopus subject areas

  • General

Cite this

Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. / Jao, Li-En; Wente, Susan R.; Chen, Wenbiao.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, No. 34, 2013, p. 13904-13909.

Research output: Contribution to journalArticle

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