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

Puping Liang; Yanwen XU; Xiya Zhang; Chenhui Ding; Rui Huang; Zhen Zhang; Jie LV; Xiaowei Xie; Yuxi Chen; Yujing LI; Ying Sun; Yaofu Bai; Zhou Songyang; Wenbin MA; Canquan Zhou; Junjiu Huang

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

Author: Puping LIANG ¹ ; Yanwen XU ¹ ; Xiya ZHANG ¹ ; Chenhui DING ¹ ; Rui HUANG ¹ ; Zhen ZHANG ¹ ; Jie LV ¹ ; Xiaowei XIE ¹ ; Yuxi CHEN ¹ ; Yujing LI ¹ ; Ying SUN ¹ ; Yaofu BAI ¹ ; Zhou SONGYANG ¹ ; Wenbin MA ¹ ; Canquan ZHOU ¹ ; Junjiu HUANG ¹
Author Information

1. Guangdong Province Key Laboratory of Reproductive Medicine, the First Affiliated Hospital, and Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275 China.
Publication Type:Journal Article
MeSH: Blastocyst; CRISPR-Cas Systems; Hemoglobins, Abnormal; genetics; metabolism; Humans; Zygote
From: Protein & Cell 2015;6(5):363-372
CountryChina
Language:English
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.