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Molecular cell

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


PMID 28431230

Abstract

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 bound to guide RNA and in complexxa0with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing andxa0pre-ordering of the seed sequence in thexa0guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals thexa0strand displacement mechanism that facilitates guide-target hybridization and suggests axa0mechanism for double-stranded DNA cleavage involving axa0single active site. Together, these insights advancexa0our mechanistic understanding of Cas12a enzymesxa0and may contribute to further development of genome editing technologies.