The crystal structure of Cpf1 in complex with CRISPR RNA

• Published in: Nature (London) 2016-04, Vol.532 (7600), p.522-526
• Main Authors: Dong, De, Ren, Kuan, Qiu, Xiaolin, Zheng, Jianlin, Guo, Minghui, Guan, Xiaoyu, Liu, Hongnan, Li, Ningning, Zhang, Bailing, Yang, Daijun, Ma, Chuang, Wang, Shuo, Wu, Dan, Ma, Yunfeng, Fan, Shilong, Wang, Jiawei, Gao, Ning, Huang, Zhiwei
• Format: Article
• Language: English
• Subjects: 631/1647/1511; 631/326/41/2536; 631/45/500; 631/535/1266; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Binding sites; Cell research; Cells; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR-Associated Proteins - chemistry; CRISPR-Associated Proteins - metabolism; CRISPR-Cas Systems; Crystal structure; Crystallography, X-Ray; Crystals; Deoxyribonucleic acid; DNA; Firmicutes - enzymology; Genetic Engineering; Genomes; Humanities and Social Sciences; letter; Models, Molecular; multidisciplinary; Nucleic Acid Conformation; Physiological aspects; Protein Binding; Protein Structure, Tertiary; Proteins; Ribonucleic acid; RNA; RNA Stability; RNA, Bacterial - chemistry; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA, Guide, CRISPR-Cas Systems - chemistry; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; Science; Structure; Substrate Specificity
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature17944

The crystal structure of monomeric Lachnospiraceae bacterium Cpf1 protein bound to CRISPR RNA is presented, establishing a framework for engineering LbCpf1 to improve its efficiency and specificity for genome editing. Cpf1 enzyme in CRISPR immunity The bacterial immune system, CRISPR, utilizes a small RNA guide, or crRNA, to target a nucleolytic CRISPR complex to DNA with a complementary sequence. This process has been widely exploited for various types of genome engineering. Previously described CRISPR systems utilize one nuclease, such as Cas6, to generate the mature crRNA, and a second, such as Cas9, to cleave the target DNA. Two studies illustrate a different approach that involves the Cpf1 protein. Emmanuelle Charpentier and colleagues report that type V-A Cpf1 protein from Francisella novicida functions as a minimalistic CRISPR system. It is a dual-nuclease enzyme that can perform both the pre-crRNA processing and DNA cleavage activities, having distinct active domains for the two substrates. Zhiwei Huang and colleagues solve the crystal structure of monomeric Lachnospiraceae bacterium Cpf1 protein bound to crRNA, showing how binding induces conformational changes in the nuclease. 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 1 , 2 , 3 , 4 , 5 , 6 , 7 . The CRISPR–Cpf1 system, a new class 2 CRISPR–Cas system, mediates robust DNA interference in human cells 1 , 8 , 9 , 10 . 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(H 2 O) 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 spec