title: CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.
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.
authors.affiliation: 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.
authors.forename: Puping
authors.initials: P
authors.lastname: Liang
authors.affiliation: 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.
authors.forename: Yanwen
authors.initials: Y
authors.lastname: Xu
authors.affiliation: 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.
authors.forename: Xiya
authors.initials: X
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Chenhui
authors.initials: C
authors.lastname: Ding
authors.affiliation: 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.
authors.forename: Rui
authors.initials: R
authors.lastname: Huang
authors.affiliation: 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.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Jie
authors.initials: J
authors.lastname: Lv
authors.affiliation: 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.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Xie
authors.affiliation: 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.
authors.forename: Yuxi
authors.initials: Y
authors.lastname: Chen
authors.affiliation: 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.
authors.forename: Yujing
authors.initials: Y
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Ying
authors.initials: Y
authors.lastname: Sun
authors.affiliation: 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.
authors.forename: Yaofu
authors.initials: Y
authors.lastname: Bai
authors.affiliation: 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.
authors.forename: Zhou
authors.initials: Z
authors.lastname: Songyang
authors.affiliation: 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.
authors.forename: Wenbin
authors.initials: W
authors.lastname: Ma
authors.affiliation: 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.
authors.forename: Canquan
authors.initials: C
authors.lastname: Zhou
authors.affiliation: 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.
authors.forename: Junjiu
authors.initials: J
authors.lastname: Huang
mesh_terms: D001755:Blastocyst; D064113:CRISPR-Cas Systems; D006455:Hemoglobins, Abnormal / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D015053:Zygote
chemical_list: D006455:Hemoglobins, Abnormal