Engineered CRISPR/Cas9 enzymes improve discrimination by slowing DNA cleavage to allow release of off-target DNA

Mu-Sen Liu, Shanzhong Gong, Helen-Hong Yu, Kyungseok Jung, Kenneth A. Johnson () and David W. Taylor ()
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Mu-Sen Liu: Department of Molecular Biosciences
Shanzhong Gong: Department of Molecular Biosciences
Helen-Hong Yu: Department of Molecular Biosciences
Kyungseok Jung: Department of Molecular Biosciences
Kenneth A. Johnson: Department of Molecular Biosciences
David W. Taylor: Department of Molecular Biosciences

Nature Communications, 2020, vol. 11, issue 1, 1-13

Abstract: Abstract CRISPR/Cas9 is a programmable genome editing tool widely used for biological applications and engineered Cas9s have increased discrimination against off-target cleavage compared with wild-type Streptococcus pyogenes (SpCas9) in vivo. To understand the basis for improved discrimination against off-target DNA containing important mismatches at the distal end of the guide RNA, we performed kinetic analyses on the high-fidelity (Cas9-HF1) and hyper-accurate (HypaCas9) engineered Cas9 variants. We show that DNA cleavage is impaired by more than 100- fold for the high-fidelity variants. The high-fidelity variants improve discrimination by slowing the observed rate of cleavage without increasing the rate of DNA rewinding and release. The kinetic partitioning favors release rather than cleavage of a bound off-target substrate only because the cleavage rate is so low. Further improvement in discrimination may require engineering increased rates of dissociation of off-target DNA.

Date: 2020
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DOI: 10.1038/s41467-020-17411-1

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