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Determinants of CRISPR Cas12a nuclease activation by DNA and RNA targets?

Nucleic Acids Research. 2024-03; 
Eric A Nalefski,?Remy M Kooistra,?Ishira Parikh,?Samantha Hedley,?Karunya Rajaraman,?Damian Madan
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摘要

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... More

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