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

Nature Communications
Mu-Sen LiuDavid W Taylor

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.

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Citations

Feb 6, 2021·Transfusion and Apheresis Science : Official Journal of the World Apheresis Association : Official Journal of the European Society for Haemapheresis·David G Justus, John P Manis
Jul 10, 2021·Scientific Reports·Trishit BanerjeeKiyoto Kamagata
Aug 13, 2021·Nature Structural & Molecular Biology·Patrick PauschJennifer A Doudna
Oct 28, 2020·Journal of Chemical Information and Modeling·Aakash SahaGiulia Palermo
Aug 16, 2021·Molecular Genetics and Metabolism·Guillermo Aquino-Jarquin

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Methods Mentioned

BETA
FRET
DNA trap

Software Mentioned

FitSpace
KinTek Explorer

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