Structure and Engineering of Francisella novicida Cas9
Author(s)Hirano, Hisato; Horii, Takuro; Kimura, Mika; Nakane, Takanori; Ishitani, Ryuichiro; Hatada, Izuho; Nishimasu, Hiroshi; Nureki, Osamu; Gootenberg, Jonathan S; Abudayyeh, Omar Osama; Hsu, Patrick; Zhang, Feng; ... Show more Show less
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Summary 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.
DepartmentMassachusetts Institute of Technology. Department of Biological Engineering; Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences; McGovern Institute for Brain Research at MIT
Hirano, Hisato et al. “Structure and Engineering of Francisella Novicida Cas9.” Cell 164, 5 (February 2016): 950–961 © 2016 Elsevier Inc
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