Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage

The structure of Cpf1, a CRISPR–Cas/RNA-guided nuclease, is presented with a three-stranded RNA–DNA loop after cleavage, providing insight into its working mechanism. The CRISPR–Cas9 system for genome editing has evolved to utilize variants of Cas9-type enzymes that possess different properties. One of these homologues is the Cpf1 enzyme, the therapeutic potential of which is starting to be explored. Guillermo Montoya and colleagues have solved the structure of Cpf1 endonuclease from Francisella novicida bound to an CRISPR RNA (crRNA)–DNA mimic of the post-cleavage complex. The new understanding of Cpf1 mechanism could be used to widen the repertoire of CRIPSR–Cas editing enzymes and their applications. Cpf1 is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool1,2. Here we provide insight into its DNA-targeting mechanism by determining the structure of Francisella novicida Cpf1 with the triple-stranded R-loop generated after DNA cleavage. The structure reveals the machinery involved in DNA unwinding to form a CRISPR RNA (crRNA)–DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix–loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner into the double-stranded DNA. Unzipping of the double-stranded DNA occurs in a cleft arranged by acidic and hydrophobic residues facilitating the crRNA–DNA hybrid formation. The PAM single-stranded DNA is funnelled towards the nuclease site through a mixed hydrophobic and basic cavity. In this catalytic conformation, the PAM-interacting domain and the helix–loop–helix motif in the REC1 domain adopt a ‘rail’ shape and ‘flap-on’ conformations, respectively, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms the ‘septum’, separating the displaced PAM strand and the crRNA–DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures3,4 proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1.