Molecular cell · 2022
The miniature CRISPR-Cas12m effector binds DNA to block transcription.
СтатьяПоддержка исследования: вне правительства СШАResearch Support, N.I.H., IntramuralНазвание и аннотация: Перевод в очереди
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Wu Wen Y, Mohanraju Prarthana, Liao Chunyu, Adiego-Pérez Belén, Creutzburg Sjoerd C A, Makarova Kira S, Keessen Karlijn, Lindeboom Timon A, Khan Tahseen S, Prinsen Stijn, Joosten Rob, Yan Winston X, Migur Anzhela, Laffeber Charlie, Scott David A, Lebbink Joyce H G, Koonin Eugene V, Beisel Chase L, van der Oost John
Аннотация
CRISPR-Cas are prokaryotic adaptive immune systems. Cas nucleases generally use CRISPR-derived RNA guides to specifically bind and cleave DNA or RNA targets. Here, we describe the experimental characterization of a bacterial CRISPR effector protein Cas12m representing subtype V-M. Despite being less than half the size of Cas12a, Cas12m catalyzes auto-processing of a crRNA guide, recognizes a 5'-TTN' protospacer-adjacent motif (PAM), and stably binds a guide-complementary double-stranded DNA (dsDNA). Cas12m has a RuvC domain with a non-canonical catalytic site and accordingly is incapable of guide-dependent cleavage of target nucleic acids. Despite lacking target cleavage activity, the high binding affinity of Cas12m to dsDNA targets allows for interference as demonstrated by its ability to protect bacteria against invading plasmids through silencing invader transcription and/or replication. Based on these molecular features, we repurposed Cas12m by fusing it to a cytidine deaminase that resulted in base editing within a distinct window.