Structure and Engineering of Francisella novicida Cas9

The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5′-NGG-3′ PAM, and used the structural information to create a variant that can recognize the more relaxed 5′-YG-3′ PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5′-YG-3′ PAM, thus expanding the target space of the CRISPR-Cas9 toolbox. [Display omitted] •Crystal structure of Francisella novicida Cas9 bound to guide RNA and target DNA•Conserved and divergent structural features among orthologous CRISPR-Cas9 systems•Rational engineering of the FnCas9-PAM specificity from 5′-NGG-3′ to 5′-YG-3′•Genome editing in mouse zygotes using pre-assembled FnCas9-sgRNA complexes The high-resolution crystal structures of Francisella novicida Cas9 reveal conserved and divergent features among distantly related CRISPR-Cas9 systems. The structural information is used to rationally design an FnCas9 variant that recognizes a more relaxed PAM sequence.