Determinants of CRISPR Cas12a nuclease activation by DNA and RNA targets

The RNA-guided CRISPR-associated (Cas) enzyme Cas12a cleaves specific double-stranded (ds-) or single-stranded (ss-) DNA targets (in cis), unleashing non-specific ssDNA cleavage (in trans). Though this trans-activity is widely coopted for diagnostics, little is known about target determinants promot...

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Bibliographic Details
Published in:	Nucleic acids research Vol. 52; no. 8; pp. 4502 - 4522
Main Authors:	Nalefski, Eric A, Kooistra, Remy M, Parikh, Ishira, Hedley, Samantha, Rajaraman, Karunya, Madan, Damian
Format:	Journal Article
Language:	English
Published:	England Oxford University Press 08-05-2024
Subjects:	Bacterial Proteins - genetics Bacterial Proteins - metabolism CRISPR-Associated Proteins - metabolism CRISPR-Cas Systems DNA - chemistry DNA - genetics DNA - metabolism DNA, Single-Stranded - metabolism Endodeoxyribonucleases - genetics Endodeoxyribonucleases - metabolism Enzyme Activation Kinetics Nucleic Acid Enzymes RNA - chemistry RNA - genetics RNA - metabolism RNA, Guide, CRISPR-Cas Systems - genetics RNA, Guide, CRISPR-Cas Systems - metabolism
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Summary:	The RNA-guided CRISPR-associated (Cas) enzyme Cas12a cleaves specific double-stranded (ds-) or single-stranded (ss-) DNA targets (in cis), unleashing non-specific ssDNA cleavage (in trans). Though this trans-activity is widely coopted for diagnostics, little is known about target determinants promoting optimal enzyme performance. Using quantitative kinetics, we show formation of activated nuclease proceeds via two steps whereby rapid binding of Cas12a ribonucleoprotein to target is followed by a slower allosteric transition. Activation does not require a canonical protospacer-adjacent motif (PAM), nor is utilization of such PAMs predictive of high trans-activity. We identify several target determinants that can profoundly impact activation times, including bases within the PAM (for ds- but not ssDNA targets) and sequences within and outside those complementary to the spacer, DNA topology, target length, presence of non-specific DNA, and ribose backbone itself, uncovering previously uncharacterized cleavage of and activation by RNA targets. The results provide insight into the mechanism of Cas12a activation, with direct implications on the role of Cas12a in bacterial immunity and for Cas-based diagnostics.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0305-1048 1362-4962
DOI:	10.1093/nar/gkae152