Hybrid Cas12a Variants with Relaxed PAM Requirements Expand Genome Editing Compatibility

Cas12a is a widely used programmable nuclease for genome editing across a variety of organisms, but its application is limited by its PAM recognition restriction. To alleviate these PAM constraints, protein engineering efforts have been applied to expand the PAM recognition range. In this study, we designed and constructed 990 synthetic hybrid Cas12a chimeras through domain shuffling and screened an efficient hybrid Cas12a (ehCas12a) that could recognize a broad range PAM of 5'-TYYN-3' (Y is T or C and N is A, T, C, or G). Furthermore, we constructed an ehCas12a variant, ehCas12a RRVR (T167R/N572R/K578V/N582R), with expanded PAM preference to 5'-TNYN, TWRV-3' (W is A or T, R is A or G, and V is A, C, or G), which can efficiently recognize -2* A/G PAMs that are barely recognized by Cas12a-type proteins and their mutants. Finally, we demonstrated that the DNase-inactivated ehCas12a RRVR base editor (dehCas12a RRVR-BE) was capable of targeting noncanonical PAMs in vivo and disease-related loci for potential therapeutic applications. Overall, our findings highlight the modular design and reconfiguration of Cas proteins for enhanced functionality.