Split Cas12a Protospacer Engineering Enables Ultraspecific Recognition of a PAM-Less DNA Strand via an External PAM-Proximal Duplex

Here, we divide the DNA activator into PAM-proximal (Pp) and PAM-distal (Pd) duplexes, spatially separating the PAM requirement from the interrogation of a PAM-less target sequence.

FRET, electrophoretic mobility shift, fluorescence polarization, and functional assays indicate that either fragment can associate with Cas12a, but that robust productive activation requires the PAM-containing Pp duplex together with the Pd duplex. Our data further support a model in which PAM-mediated protein–DNA contacts stabilize an assembly-competent complex and facilitate binding of the PAM-less Pd target capture.