Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d

• Published in: Nature communications Vol. 10; no. 1; p. 2544
• Main Authors: Zhang, Bo, Ye, Yangmiao, Ye, Weiwei, Perčulija, Vanja, Jiang, Han, Chen, Yiyang, Li, Yu, Chen, Jing, Lin, Jinying, Wang, Siqi, Chen, Qi, Han, Yu-San, Ouyang, Songying
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 11.06.2019; Nature Publishing Group; Nature Portfolio
• Subjects: 631/250; 631/337/4041; 631/45/535/1266; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cleavage; Clustered Regularly Interspaced Short Palindromic Repeats; Conformation; CRISPR; CRISPR-Associated Proteins - chemistry; CRISPR-Associated Proteins - genetics; CRISPR-Associated Proteins - metabolism; CRISPR-Cas Systems; Domains; Functional analysis; Humanities and Social Sciences; Magnesium; Maturation; Metal ions; multidisciplinary; Nucleic Acid Conformation; Nucleotide sequence; Nucleotides; Protein Domains; Ribonuclease; Ribonucleases - chemistry; Ribonucleases - genetics; Ribonucleases - metabolism; Ribonucleic acid; RNA; RNA Processing, Post-Transcriptional; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA, Guide, CRISPR-Cas Systems - genetics; Ruminococcus - enzymology; Ruminococcus - genetics; Science; Science (multidisciplinary); Spacer; Structure-function relationships
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-019-10507-3

Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. Here we report the detailed structural and functional analysis of the uncultured Ruminococcus sp . Cas13d (UrCas13d)-crRNA complex. Two hydrated Mg 2+ ions aid in stabilizing the conformation of the crRNA repeat region. Sequestration of divalent metal ions does not alter pre-crRNA processing, but abolishes target cleavage by UrCas13d. Notably, the pre-crRNA processing is executed by the HEPN-2 domain. Furthermore, both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are specifically recognized by UrCas13d. Moreover, correct base pairings within two separate spacer regions (an internal and a 3′-end region) are essential for target cleavage. These findings provide a framework for the development of Cas13d into a tool for a wide range of applications. Cas13d is a class 2 type VI-D CRISPR-Cas RNA-guided RNase. Here the authors present the high-resolution crystal structure of the uncultured Ruminococcus sp . Cas13d (UrCas13d)-crRNA complex and by combining structural, mutational and biochemical studies provide mechanistic insights into the CRISPR-Cas13d system.