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Nucleic Acids Res ; 52(8): 4502-4522, 2024 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-38477377

RESUMEN

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.


Asunto(s)
Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , ADN de Cadena Simple , Endodesoxirribonucleasas , ARN , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/metabolismo , ADN/metabolismo , ADN/genética , ADN/química , ADN de Cadena Simple/metabolismo , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Activación Enzimática , Cinética , ARN/metabolismo , ARN/química , ARN/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética
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