Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus

被引：145

作者：

Bothmer, Anne ^{[1
]}

Phadke, Tanushree ^{[1
]}

Barrera, Luis A. ^{[1
]}

Margulies, Carrie M. ^{[1
]}

Lee, Christina S. ^{[1
]}

Buquicchio, Frank ^{[1
]}

Moss, Sean ^{[1
]}

Abdulkerim, Hayat S. ^{[1
]}

Selleck, William ^{[1
]}

Jayaram, Hariharan ^{[1
]}

Myer, Vic E. ^{[1
]}

Cotta-Ramusino, Cecilia ^{[1
]}

机构：

[1] Editas Med, 11 Hurley St, Cambridge, MA 02141 USA

来源：

NATURE COMMUNICATIONS | 2017年 / 8卷

关键词：

STRAND BREAK REPAIR; REPLICATION FORK REVERSAL; HOMOLOGOUS RECOMBINATION; CAS9; NUCLEASES; RNA; SPECIFICITY; END; IMMUNITY; CELLS; CLEAVAGE;

D O I：

10.1038/ncomms13905

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

The CRISPR-Cas9 system provides a versatile toolkit for genome engineering that can introduce various DNA lesions at specific genomic locations. However, a better understanding of the nature of these lesions and the repair pathways engaged is critical to realizing the full potential of this technology. Here we characterize the different lesions arising from each Cas9 variant and the resulting repair pathway engagement. We demonstrate that the presence and polarity of the overhang structure is a critical determinant of double-strand break repair pathway choice. Similarly, single nicks deriving from different Cas9 variants differentially activate repair: D10A but not N863A-induced nicks are repaired by homologous recombination. Finally, we demonstrate that homologous recombination is required for repairing lesions using double-stranded, but not single-stranded DNA as a template. This detailed characterization of repair pathway choice in response to CRISPR-Cas9 enables a more deterministic approach for designing research and therapeutic genome engineering strategies.

引用

页数：12

共 44 条

[1] Alternative-NHEJ Is a Mechanistically Distinct Pathway of Mammalian Chromosome Break Repair [J].