Efficient Multiple Genome Modifications Induced by the crRNAs, tracrRNA and Cas9 Protein Complex in Zebrafish.

Hirohito Kotani,Rie Ohga,Atsuo Kawahara,Satoshi Ota,Kiyohito Taimatsu,Hodaka Fujii

doi:10.1371/journal.pone.0128319

Abstract

The type II clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas9 endonuclease (CRISPR/Cas9) has become a powerful genetic tool for understanding the function of a gene of interest. In zebrafish, the injection of Cas9 mRNA and guide-RNA (gRNA), which are prepared using an in vitro transcription system, efficiently induce DNA double-strand breaks (DSBs) at the targeted genomic locus. Because gRNA was originally constructed by fusing two short RNAs CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), we examined the effect of synthetic crRNAs and tracrRNA with Cas9 mRNA or Cas9 protein on the genome editing activity. We previously reported that the disruption of tyrosinase (tyr) by tyr-gRNA/Cas9 mRNA causes a retinal pigment defect, whereas the disruption of spns2 by spns2-gRNA1/Cas9 mRNA leads to a cardiac progenitor migration defect in zebrafish. Here, we found that the injection of spns2-crRNA1, tyr-crRNA and tracrRNA with Cas9 mRNA or Cas9 protein simultaneously caused a migration defect in cardiac progenitors and a pigment defect in retinal epithelial cells. A time course analysis demonstrated that the injection of crRNAs and tracrRNA with Cas9 protein rapidly induced genome modifications compared with the injection of crRNAs and tracrRNA with Cas9 mRNA. We further show that the crRNA-tracrRNA-Cas9 protein complex is functional for the visualization of endogenous gene expression; therefore, this is a very powerful, ready-to-use system in zebrafish.

Highlights

Recent remarkable innovations in genome editing technologies, such as transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, enable us to induce genome modifications at targeted genomic loci [1,2,3,4,5,6,7]
The recent evidence suggests that the injection of guide RNA (gRNA) and Cas9 mRNA into fertilized embryos efficiently induces double-strand breaks (DSBs) at the targeted genomic locus in various model organisms [31,32]
When two CRISPR RNA (crRNA) and tracrRNA with the Cas9 protein were injected into zebrafish embryos, the two expected phenotypes such as cardia bifida and an eye pigment defect were observed with high efficiency (Table 1)

Summary

Introduction

Recent remarkable innovations in genome editing technologies, such as transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, enable us to induce genome modifications at targeted genomic loci [1,2,3,4,5,6,7]. These systems allow us to generate loss-offunction alleles by frameshift-mediated mutations and establish knock-in alleles using donor DNA in various model organisms, including zebrafish [8,9,10,11,12]. Two groups have reported the efficient CRISPR/Cas9-mediated knock-in of a donor DNA by the homology-independent DSBs repair system [20,21]

Methods

Results

Conclusion