Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA.
Cpf1 is an RNA-guided endonuclease of a type V CRISPR-Cas system that has been recently harnessed for genome editing. Here, we report the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with the guide RNA and its target DNA at 2.8 Å resolution. AsCpf1 adopts a bilobed architecture, with the RNA-DNA heteroduplex bound inside the central channel. The structural comparison of AsCpf1 with Cas9, a type II CRISPR-Cas nuclease, reveals both striking similarity and major differences, thereby explaining their distinct functionalities. AsCpf1 contains the RuvC domain and a putative novel nuclease domain, which are responsible for cleaving the non-target and target strands, respectively, and for jointly generating staggered DNA double-strand breaks. AsCpf1 recognizes the 5'-TTTN-3' protospacer adjacent motif by base and shape readout mechanisms. Our findings provide mechanistic insights into RNA-guided DNA cleavage by Cpf1 and establish a framework for rational engineering of the CRISPR-Cpf1 toolbox.
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan.
Takashi
T
Yamano
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan; JST, PRESTO, Tokyo 113-0032, Japan.
Hiroshi
H
Nishimasu
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Developmental Pathology, Institute of Pathology, Bonn Medical School, 53127 Bonn, Germany.
Bernd
B
Zetsche
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan.
Hisato
H
Hirano
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Ian M
IM
Slaymaker
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Yinqing
Y
Li
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia; Skolkovo Institute of Science and Technology, Skolkovo, 143026, Russia.
Iana
I
Fedorova
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan.
Takanori
T
Nakane
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
Kira S
KS
Makarova
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
Eugene V
EV
Koonin
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan.
Ryuichiro
R
Ishitani
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: zhang@broadinstitute.org.
Feng
F
Zhang
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan. Electronic address: nureki@bs.s.u-tokyo.ac.jp.
Osamu
O
Nureki
D045850:Acidaminococcus / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D005821:Genetic Techniques; D008958:Models, Molecular; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D009692:Nucleic Acid Heteroduplexes; D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA