CRISPR RNA-independent activation of Cas12a

CRISPR-Cas12a nucleases are classically activated through CRISPR RNA (crRNA)-guided and PAM-dependent target recognition, which together establish a canonical heteroduplex associated with nuclease activation. Here we identify a crRNA– and PAM-independent activation pathway for Cas12a that reveals previously unrecognized conformational plasticity within its nucleic acid–recognition interface. We show that short RNAs can directly occupy the canonical crRNA–binding channel and trigger a catalytically competent trans cleavage state in the absence of PAM recognition or canonical R-loop formation. Biochemical assays indicate that short RNAs bind the crRNA–binding channel and are competitively displaced by cognate crRNA, consistent with binding at a conserved nucleic acid–binding interface. Cryo-electron microscopy (cryo-EM) further reveals that Cas12a maintains its global catalytic architecture while exhibiting loss of canonical PAM-dependent stabilization and increased flexibility of the RuvC lid, alongside accommodation of a noncanonical RNA–DNA hybrid with inverted polarity relative to the crRNA–target duplex. This crRNA-independent activation pathway enables programmable and amplification-free detection of DNA and RNA targets independent of canonical guide-mediated recognition. Together, these findings define an alternative activation geometry for Cas12a and expand models of Class 2 CRISPR-Cas effector activation beyond crRNA– and PAM-directed recognition.