The crystal structure of Cpf1 in complex with CRISPR RNA.
The CRISPR-Cas systems, as exemplified by CRISPR-Cas9, are RNA-guided adaptive immune systems used by bacteria and archaea to defend against viral infection. The CRISPR-Cpf1 system, a new class 2 CRISPR-Cas system, mediates robust DNA interference in human cells. Although functionally conserved, Cpf1 and Cas9 differ in many aspects including their guide RNAs and substrate specificity. Here we report the 2.38 Å crystal structure of the CRISPR RNA (crRNA)-bound Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1). LbCpf1 has a triangle-shaped architecture with a large positively charged channel at the centre. Recognized by the oligonucleotide-binding domain of LbCpf1, the crRNA adopts a highly distorted conformation stabilized by extensive intramolecular interactions and the (Mg(H2O)6)(2+) ion. The oligonucleotide-binding domain also harbours a looped-out helical domain that is important for LbCpf1 substrate binding. Binding of crRNA or crRNA lacking the guide sequence induces marked conformational changes but no oligomerization of LbCpf1. Our study reveals the crRNA recognition mechanism and provides insight into crRNA-guided substrate binding of LbCpf1, establishing a framework for engineering LbCpf1 to improve its efficiency and specificity for genome editing.
School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
De
D
Dong
School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
Kuan
K
Ren
School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
Xiaolin
X
Qiu
School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
Jianlin
J
Zheng
School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
Minghui
M
Guo
School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
Xiaoyu
X
Guan
School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
Hongnan
H
Liu
Ministry of Education Key Laboratory of Protein Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
Ningning
N
Li
School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
Bailing
B
Zhang
School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
Daijun
D
Yang
School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
Chuang
C
Ma
School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
Shuo
S
Wang
School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
Dan
D
Wu
School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
Yunfeng
Y
Ma
Ministry of Education Key Laboratory of Protein Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
Shilong
S
Fan
Ministry of Education Key Laboratory of Protein Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
Jiawei
J
Wang
Ministry of Education Key Laboratory of Protein Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
Ning
N
Gao
School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
Zhiwei
Z
Huang
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D064130:CRISPR-Associated Proteins / Q000737:chemistry* / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D018360:Crystallography, X-Ray; D000068536:Bacillota / Q000201:enzymology*; D005818:Genetic Engineering; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020871:RNA Stability; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D012329:RNA, Bacterial; D000094704:RNA, Guide, CRISPR-Cas Systems