Trans-cleavage activity of Cas12a effectors can be unleashed by both double-stranded DNA and single-stranded RNA targeting in absence of PAM.

CRISPR-Cas12a is a powerful tool in nucleic acid detection, but the relationship between its trans-cleavage activity and protospacer adjacent motif (PAM) sequences remains incompletely understood. In this study, we synthesized diverse PAM-sequence substrates and conducted systematic cis-cleavage and trans-cleavage experiments with three Cas12a orthologs. We found that double-stranded DNA (dsDNA) can activate Cas12a's trans-cleavage activity even without PAM and this activation occurring independently of cis-cleavage. Notably, our results also revealed that single-stranded RNA (ssRNA) can directly initiate the trans-cleavage activity of Cas12a.We also experimentally validated the feasibility of CRISPR-Cas12a in detecting target dsDNA lacking PAM sequences, including identifying mutated sites in clinical samples. Structural prediction using AlphaFold 3 revealed the potential mechanism of Cas12a's PAM-independent trans-cleavage. Our research expands the understanding of Cas12a's trans-cleavage mechanism and demonstrates its potential for nucleic acid detection beyond PAM-dependent targets. This discovery broadens the application scope of Cas12a, providing new opportunities for developing highly sensitive and versatile diagnostic platforms.