Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining

被引:7
作者
Feng, Yi-Li [1 ,2 ,3 ]
Liu, Si-Cheng [1 ,2 ,3 ]
Chen, Ruo-Dan [1 ,2 ,3 ]
Sun, Xiu-Na [1 ,2 ,3 ]
Xiao, Jing-Jing [1 ,2 ,3 ]
Xiang, Ji-Feng [1 ,2 ,3 ,4 ]
Xie, An-Yong [1 ,2 ,3 ]
机构
[1] Zhejiang Univ, Sir Run Run Shaw Hosp, Innovat Ctr Minimally Invas Tech & Device, Dept Gen Surg,Sch Med, Hangzhou, Zhejiang, Peoples R China
[2] Zhejiang Univ, Inst Translat Med, Sch Med, Hangzhou, Peoples R China
[3] Zhejiang Univ, Canc Ctr, Hangzhou, Zhejiang, Peoples R China
[4] Chongqing Gen Hosp, Dept Gen Surg, Chongqing 400013, Peoples R China
基金
中国国家自然科学基金;
关键词
DISTINCT ROLES; CRISPR; ENDONUCLEASE; PROTEIN; RECOMBINATION; XRCC4; INTERROGATION; EFFICIENCY; NUCLEASES; KU80;
D O I
10.1093/nar/gkad116
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
In CRISPR/Cas9 genome editing, the tight and persistent target binding of Cas9 provides an opportunity for efficient genetic and epigenetic modification on genome. In particular, technologies based on catalytically dead Cas9 (dCas9) have been developed to enable genomic regulation and live imaging in a site-specific manner. While post-cleavage target residence of CRISPR/Cas9 could alter the pathway choice in repair of Cas9-induced DNA double strand breaks (DSBs), it is possible that dCas9 residing adjacent to a break may also determine the repair pathway for this DSB, providing an opportunity to control genome editing. Here, we found that loading dCas9 onto a DSB-adjacent site stimulated homology-directed repair (HDR) of this DSB by locally blocking recruitment of classical non-homologous end-joining (c-NHEJ) factors and suppressing c-NHEJ in mammalian cells. We further repurposed dCas9 proximal binding to increase HDR-mediated CRISPR genome editing by up to 4-fold while avoiding exacerbation of off-target effects. This dCas9-based local inhibitor provided a novel strategy of c-NHEJ inhibition in CRISPR genome editing in place of small molecule c-NHEJ inhibitors, which are often used to increase HDR-mediated genome editing but undesirably exacerbate off-target effects.
引用
收藏
页码:2740 / 2758
页数:19
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