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Cell Discov. 2018 Jul 24;4:46. doi: 10.1038/s41421-018-0049-7. eCollection 2018.

Programmable DNA repair with CRISPRa/i enhanced homology-directed repair efficiency with a single Cas9.

Ye L#1,2,3, Wang C#1, Hong L1,4, Sun N1, Chen D1, Chen S2,3, Han F1.

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1Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, 310058 Hangzhou, China.
System Biology Institute, Integrated Science & Technology Center, 850 West Campus Drive, Room 361, West Haven, CT 06516 USA.
3Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510 USA.
4Cardiovascular Research Center, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520 USA.
Contributed equally


CRISPR systems have been proven as versatile tools for site-specific genome engineering in mammalian species. During the gene editing processes, these RNA-guide nucleases introduce DNA double strand breaks (DSBs), in which non-homologous DNA end joining (NHEJ) dominates the DNA repair pathway, limiting the efficiency of homology-directed repair (HDR), the alternative pathway essential for precise gene targeting. Multiple approaches have been developed to enhance HDR, including chemical compound or RNA interference-mediated inhibition of NHEJ factors, small molecule activation of HDR enzymes, or cell cycle timed delivery of CRISPR complex. However, these approaches face multiple challenges, yet have moderate or variable effects. Here we developed a new approach that programs both NHEJ and HDR pathways with CRISPR activation and interference (CRISPRa/i) to achieve significantly enhanced HDR efficiency of CRISPR-mediated gene editing. The manipulation of NHEJ and HDR pathway components, such as CtIP, CDK1, KU70, KU80, and LIG4, was mediated by catalytically dead guide RNAs (dgRNAs), thus relying on only a single catalytically active Cas9 to perform both CRISPRa/i and precise gene editing. While reprogramming of most DNA repair factors or their combinations tested enhanced HDR efficiency, simultaneously activating CDK1 and repressing KU80 has the strongest effect with increased HDR rate upto an order of magnitude. Doxycycline-induced dgRNA-based CRISPRa/i programming of DNA repair enzymes, as well as viral packaging enabled flexible and tunable HDR enhancement for broader applicability in mammalian cells. Our study provides an effective, flexible, and potentially safer strategy to enhance precise genome modifications, which might broadly impact human gene editing and therapy.

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