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- W3129126865 abstract "Cas12a is an RNA-guided DNA endonuclease of the type V-A CRISPR-Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity in Streptococcus pyogenes (Spy) Cas9. Here, we examined a BH variant of Cas12a from Francisella novicida (FnoCas12aKD2P ) to test mechanistic conservation. Our results show that for RNA-guided DNA cleavage (cis-activity), FnoCas12aKD2P accumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12aKD2P also possess reduced trans-single-stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in both cis- and trans-cleavages and show its conserved role in target discrimination among Cas nucleases." @default.
- W3129126865 created "2021-02-15" @default.
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- W3129126865 date "2021-02-28" @default.
- W3129126865 modified "2023-10-17" @default.
- W3129126865 title "The bridge helix of Cas12a imparts selectivity in <i>cis</i> ‐DNA cleavage and regulates <i>trans</i> ‐DNA cleavage" @default.
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- W3129126865 doi "https://doi.org/10.1002/1873-3468.14051" @default.
- W3129126865 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/8044059" @default.
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- W3129126865 hasPublicationYear "2021" @default.
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