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The bridge helix of Cas12a imparts selectivity in cis ‐DNA cleavage and regulates trans ‐DNA cleavage
Author(s) -
Parameshwaran Hari Priya,
Babu Kesavan,
Tran Christine,
Guan Kevin,
Allen Aleique,
Kathiresan Venkatesan,
Qin Peter Z.,
Rajan Rakhi
Publication year - 2021
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1002/1873-3468.14051
Subject(s) - dna , cleavage (geology) , endonuclease , cas9 , crispr , dna supercoil , rna , chemistry , biology , microbiology and biotechnology , biochemistry , gene , dna replication , paleontology , fracture (geology)
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 (FnoCas12a KD2P ) to test mechanistic conservation. Our results show that for RNA‐guided DNA cleavage ( cis‐ activity), FnoCas12a KD2P accumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12a KD2P 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.