Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants

dc.contributor.author Seetharam, Arun
dc.contributor.author Murugan, Karthik
dc.contributor.author Suresh, Shravanti
dc.contributor.author Seetharam, Arun
dc.contributor.author Severin, Andrew
dc.contributor.author Sashital, Dipali
dc.contributor.author Severin, Andrew
dc.contributor.department Office of Biotechnology
dc.contributor.department Biochemistry, Biophysics and Molecular Biology
dc.contributor.department Genome Informatics Facility
dc.contributor.department Molecular, Cellular and Developmental Biology
dc.date 2021-03-30T19:56:09.000
dc.date.accessioned 2021-04-29T23:59:27Z
dc.date.available 2021-04-29T23:59:27Z
dc.date.copyright Fri Jan 01 00:00:00 UTC 2021
dc.date.issued 2021-03-21
dc.description.abstract <p>Cas9 is an RNA-guided endonuclease in the bacterial CRISPR–Cas immune system and a popular tool for genome editing. The commonly used <em>Streptococcus pyogenes</em> 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 <em>in vitro</em> cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and <em>Staphylococcus aureus</em> 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.</p>
dc.description.comments <p>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." <em>Nucleic Acids Research</em> (2021). doi: <a href="https://doi.org/10.1093/nar/gkab163">10.1093/nar/gkab163</a>.</p>
dc.format.mimetype application/pdf
dc.identifier archive/lib.dr.iastate.edu/biotech_pubs/10/
dc.identifier.articleid 1009
dc.identifier.contextkey 22248241
dc.identifier.s3bucket isulib-bepress-aws-west
dc.identifier.submissionpath biotech_pubs/10
dc.identifier.uri https://dr.lib.iastate.edu/handle/20.500.12876/104642
dc.language.iso en
dc.source.bitstream archive/lib.dr.iastate.edu/biotech_pubs/10/2021_Severin_SystematicSpecificity.pdf|||Fri Jan 14 18:11:12 UTC 2022
dc.source.uri 10.1093/nar/gkab163
dc.subject.disciplines Biochemistry, Biophysics, and Structural Biology
dc.subject.disciplines Biotechnology
dc.subject.disciplines Genetics and Genomics
dc.title Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants
dc.type article
dc.type.genre article
dspace.entity.type Publication
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