The miniature CRISPR-Cas12m effector binds DNA to block transcription

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...

Full description

Saved in:

Bibliographic Details
Published in:	Molecular cell Vol. 82; no. 23; pp. 4487 - 4502.e7
Main Authors:	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
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01-12-2022
Subjects:	base editing Cas12m CRISPR-Associated Proteins - metabolism CRISPR-Cas CRISPR-Cas Systems DNA - genetics gene silencing miniature Cas Plasmids RNA RNA, Guide, CRISPR-Cas Systems - metabolism type V-M CRISPR-Cas type V-M gene silencing miniature Cas base editing Cas12m
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	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. [Display omitted] •Type V-M is an additional subtype of type V CRISPR-Cas system (Cas12-U1)•The miniature Cas12m lacks target-specific as well as collateral nuclease activity•Guide-specific silencing by Cas12m occurs through dsDNA binding•A synthetic Cas12m-cytidine deaminase has a distinct C-to-T base editing window In this work, Wu et al. describe the characterization of a small CRISPR effector protein Cas12m representing subtype V-M. Cas12m is guided by a single crRNA to stably bind but not cleave complementary double-stranded DNA, potentially contributing to host defense by silencing the expression of invader DNA.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1097-2765 1097-4164
DOI:	10.1016/j.molcel.2022.11.003