Basis for discrimination by engineered CRISPR/Cas9 enzymes

Liu, M.-S.; Gong, S.; Yu, H.-H.; Jung, K.; Johnson, K. A.; Taylor, D. W.

doi:10.1101/630509

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bioRxiv · 10.1101/630509

Basis for discrimination by engineered CRISPR/Cas9 enzymes

Liu, M.-S.·Gong, S.·Yu, H.-H.·Jung, K.·Johnson, K. A.·Taylor, D. W.

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Abstract

CRISPR/Cas9 is a programmable genome editing tool that has been widely used for biological applications. While engineered Cas9s have been reported to increase discrimination against off-target cleavage compared with wild type Streptococcus pyogenes (SpCas9) in vivo, the mechanism for enhanced specificity has not been extensively characterized. 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. While DNA unwinding is the rate-limiting step for on-target cleavage by SpCas9, we show that chemistry is seriously impaired by more than 100-fold for the high-fidelity variants. The high-fidelity variants improve discrimination by slowing the rate of chemistry without increasing the rate of DNA rewinding--the kinetic partitioning favors release rather than cleavage of a bound off-target substrate because chemistry is slow. Further improvement in discrimination may require engineering increased rates of dissociation of off-target DNA.

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Liu, M.-S., Gong, S., Yu, H.-H., Jung, K., Johnson, K. A., Taylor, D. W.. 2019-05-08. Basis for discrimination by engineered CRISPR/Cas9 enzymes. https://doi.org/10.1101/630509

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