Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish

Yu Hisano,Shota Nakade,Atsuo Kawahara,Rie Ohga,Takashi Yamamoto,Satoshi Ota,Hitoshi Okamoto,Tetsushi Sakuma

doi:10.1038/srep08841

Abstract

The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10–40 bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes (tyrosinase and krtt1c19e) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish.

Highlights

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas[9] system provides a powerful tool for genome editing in various model organisms, including zebrafish
Introduction of short homology arms into the donor vector leads to the precise integration of exogenous DNA into the zebrafish genome
We chose tyrosinase/tyr as a targeted genomic locus because the disrupted phenotype of tyr is well known as a loss of pigmentation and we had succeeded in designing a good guide RNA (gRNA), inducing indel mutations at a rate of 79%25

Summary

Introduction

The CRISPR/Cas[9] system provides a powerful tool for genome editing in various model organisms, including zebrafish. A rtificial site-specific nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like nucleases (TALENs) and RNA-guided nucleases (RGNs) have become fundamental tools for genome editing in various model organisms, including zebrafish[1,2,3,4,5] Both ZFNs and TALENs are chimeric proteins fusing the DNA-recognising domain and the FokI nuclease catalytic domain, and they function as dimers. A novel method capable of integrating long exogenous DNA fragments into the genome at high frequency was reported[21,22,23] In this method, using a donor vector containing recognition sequences of artificial site-specific nucleases, a targeted genomic locus and a donor vector are simultaneously cleaved and connected to each other, presumably through NHEJ, resulting in the integration of the entire donor vector into the targeted genomic locus. We report the efficient and precise integration of exogenous DNA into the zebrafish genome using a donor vector harbouring gRNA target sequences and short homologous sequences flanking the genomic target locus

Methods

Results

Conclusion