Unleashing high trans-substrate cleavage kinetics of Cas12a for nucleic acid diagnostics.

ABSTRACT: CRISPR (clustered regularly interspaced short palindromic repeats)-based nucleic acid diagnostics enable rapid, sensitive pathogen detection. Cas12a is frequently used in these assays because target-activated trans cleavage of a reporter molecule generates an easily detectable signal. However, variable activity across assays suggests that the catalytic potential of Cas12a has been limited via unknown mechanisms. Here, we show that Cas12a trans-nuclease activity is auto-inhibited by long PAM-proximal DNA (>120 bp) following cis-cleavage of targets. Short targets (<100 bp), optimized trans cleavage substrates, and low salt buffers unleash high catalytic efficiency (≈108 M-1 s-1) and turnover (≈1 s-1) across Cas12a orthologs. Pooling multiple Cas12a ribonucleoproteins (RNPs) targeting cluster

SUBMITTER: Nalefski EA 

PROVIDER: S-EPMC12604663 | biostudies-literature | 2025 Jul

REPOSITORIES: biostudies-literature