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Unified energetics analysis unravels SpCas9 cleavage activity for optimal gRNA design.

Authors :
Dong Zhang, Shahrouz
Hurst, Travis
Dongsheng Duan
Shi-Jie Chen
Source :
Proceedings of the National Academy of Sciences of the United States of America; 4/30/2019, Vol. 116 Issue 18, p8639-8698, 6p
Publication Year :
2019

Abstract

While CRISPR/Cas9 is a powerful tool in genome engineering, the ontarget activity and off-target effects of the system widely vary because of the differences in guide RNA (gRNA) sequences and genomic environments. Traditional approaches rely on separate models and parameters to treat on- and off-target cleavage activities. Here, we demonstrate that a free-energy scheme dominates the Cas9 editing efficacy and delineate a method that simultaneously considers on-target activities and off-target effects. While data-driven machinelearning approaches learn rules to model particular datasets, they may not be as transferrable to new systems or capable of producing new mechanistic insights as principled physical approaches. By integrating the energetics of R-loop formation under Cas9 binding, the effect of the protospacer adjacent motif sequence, and the folding stability of the whole single guide RNA, we devised a unified, physical model that can apply to any cleavage-activity dataset. This unified framework improves predictions for both on-target activities and offtarget efficiencies of spCas9 and may be readily transferred to other systems with different guide RNAs or Cas9 ortholog proteins. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
00278424
Volume :
116
Issue :
18
Database :
Complementary Index
Journal :
Proceedings of the National Academy of Sciences of the United States of America
Publication Type :
Academic Journal
Accession number :
136214401
Full Text :
https://doi.org/10.1073/pnas.1820523116