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Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption

Title
Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption
Author
Ramakrishna Suresh
Keywords
RNA; SPECIFICITY; CELLS; GUIDE; ENRICHMENT; REPAIR
Issue Date
2018-03
Publisher
OXFORD UNIV PRESS
Citation
NUCLEIC ACIDS RESEARCH, v. 46, no. 12, Article no. e71
Abstract
The use of paired Cas9 nickases instead of Cas9 nuclease drastically reduces off-target effects. Because both nickases must function for a nickase pair to make a double-strand break, the efficiency of paired nickases can intuitively be expected to be lower than that of either corresponding nuclease alone. Here, we carefully compared the gene-disrupting efficiency of Cas9 paired nickases with that of nucleases. Interestingly, the T7E1 assay and deep sequencing showed that on-target efficiency of paired D10A Cas9 nickases was frequently comparable, but sometimes higher than that of either corresponding nucleases in mammalian cells. As the underlying mechanism, we found that the HNH domain, which is preserved in the D10A Cas9 nickase, has higher activity than the RuvC domain in mammalian cells. In this study, we showed: (i) the in vivo cleavage efficiency of the HNH domain of Cas9 in mammalian cells is higher than that of the RuvC domain, (ii) paired Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption. We envision that our findings which were overlooked in previous reports will serve as a new potential guideline for tool selection for CRISPR-Cas9-mediated gene disruption, facilitating efficient and precise genome editing.
URI
https://academic.oup.com/nar/article/46/12/e71/4951847https://repository.hanyang.ac.kr/handle/20.500.11754/117804
ISSN
0305-1048; 1362-4962
DOI
10.1093/nar/gky222
Appears in Collections:
GRADUATE SCHOOL OF BIOMEDICAL SCIENCE AND ENGINEERING[S](의생명공학전문대학원) > ETC
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