Campus Units

Biochemistry, Biophysics and Molecular Biology, Office of Biotechnology, Genome Informatics Facility, Molecular, Cellular and Developmental Biology

Document Type

Article

Publication Version

Published Version

Publication Date

3-21-2021

Journal or Book Title

Nucleic Acids Research

DOI

10.1093/nar/gkab163

Abstract

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR–Cas immune system and a popular tool for genome editing. The commonly used 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. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically 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 could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.

Comments

This article is published as Murugan, Karthik, Shravanti K. Suresh, Arun S. Seetharam, Andrew J. Severin, and Dipali G. Sashital. "Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants." Nucleic Acids Research (2021). doi: 10.1093/nar/gkab163.

Creative Commons License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

Copyright Owner

The Author(s)

Language

en

File Format

application/pdf

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