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bioRxiv · 10.1101/2020.05.12.091991

High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

Murugan, K.·Seetharam, A. S.·Severin, A. J.·Sashital, D. G.
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Abstract

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The most commonly used Cas9 variant, Streptococcus pyogenes Cas9 (SpCas9), is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific than wild-type (WT) SpCas9. However, systematic comparisons of the cleavage activities of these Cas9 variants have not been reported. In this study, we employed our high-throughput in vitro cleavage assay to compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9, and HiFi Cas9). We observed that all Cas9s tested were able to cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on the target sequence and Cas9 variant. For targets with multiple mismatches, SaCas9 and engineered SpCas9 variants are more prone to nicking, while WT SpCas9 creates double-strand breaks (DSB). These differences in cleavage rates and DSB formation may account for the varied specificities observed in genome editing studies. Our analysis reveals mismatch position-dependent, off-target nicking activity of Cas9 variants which have been underreported in previous in vivo studies.

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Murugan, K., Seetharam, A. S., Severin, A. J., Sashital, D. G.. 2020-05-12. High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants. https://doi.org/10.1101/2020.05.12.091991

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