Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a

The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs, and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a in a binary complex with a guide RNA, and in a R-loop complex containing a non-cleavable guide RNA precursor and full-length target DNA. Corroborated by biochemical experiments, these structures elucidate the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. The R-loop complex structure furthermore reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes that may contribute to further development of genome editing technologies.