Determinants of CRISPR Cas12a nuclease activation by DNA and RNA targets

Abstract

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.

DNAzyme-activated CRISPR/Cas assay for sensitive and one-pot detection of lead contamination

“…[14][15][16][17] Among them, the CRISPR/Cas12a system exhibits a high catalytic efficiency with a k cat /K m value ranging from 10 6 to 10 9 s À1 M À1 . [18][19][20] Harnessing the exceptional catalytic efficiency, the CRISPR/Cas12a system promises to increase the sensitivity of DNAzyme-based assays. DNAzyme-CRISPR tandem assays, while promising in bypassing nucleic acid amplification, 4,21 often encounter challenges due to the necessity of a separation step, [22][23][24] making the assays incapable of homogeneous reaction in one tube and less ideal for on-site detection.…”