CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.

Puping Liang,Xiaowei Xie,Zhou Songyang,Wenbin Ma,Rui Huang,Junjiu Huang,Yujing Li,Yanwen Xu,Xiya Zhang,Canquan Zhou,Chenhui Ding,Zhen Zhang,Jie Lv,Yaofu Bai,Ying Sun,Yuxi Chen

doi:10.1007/s13238-015-0153-5

Puping Liang, Xiaowei Xie + Show 14 more

Open Access

https://doi.org/10.1007/s13238-015-0153-5

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Journal: Protein & cell	Publication Date: Apr 18, 2015
Citations: 941	License type: CC BY 4.0

Affiliation: Sun Yat-sen University

Abstract

Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0153-5) contains supplementary material, which is available to authorized users.

Highlights

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 RNA-endonuclease complex, consisting of the Cas9 protein and the guide RNA (∼99 nt), is based on the adaptive immune system of streptococcus pyogenes SF370
Three groups recently found through whole genome sequencing that off-target effects of CRISPR/Cas9 appeared rare in human pluripotent stem cells (Smith et al, 2014b; Suzuki et al, 2014; Veres et al, 2014), raising the possibility that high frequencies of unintended targeting by CRISPR/Cas9 may be more prevalent in cancer cell lines
We report that the CRISPR/Cas9 system can cleave endogenous gene efficiently in human tripronuclear zygotes, and that the double strand break (DSB) generated by CRISPR/Cas9 cleavage are repaired by non-homologous end joining (NHEJ) and homologous recombination directed repair (HDR)

Summary

Introduction

The CRISPR/Cas RNA-endonuclease complex, consisting of the Cas protein and the guide RNA (gRNA) (∼99 nt), is based on the adaptive immune system of streptococcus pyogenes SF370 It targets genomic sequences containing the tri-nucleotide protospacer adjacent motif (PAM) and complementary to the gRNA, and can be programmed to recognize virtually any genes through the manipulation of gRNA sequences (Cho et al, 2013; Cong et al, 2013; Jinek et al, 2012; Jinek et al, 2013; Mali et al, 2013c). Because ethical concerns preclude studies of gene editing in normal embryos, we decided to use tripronuclear (3PN) zygotes, which have one oocyte nucleus and two sperm nuclei

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