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Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d
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Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d
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Journal Article
Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d
2019
Overview
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.
Publisher
Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
Subject
/ Bacterial Proteins - chemistry
/ Bacterial Proteins - genetics
/ Bacterial Proteins - metabolism
/ Cleavage
/ Clustered Regularly Interspaced Short Palindromic Repeats
/ CRISPR
/ CRISPR-Associated Proteins - chemistry
/ CRISPR-Associated Proteins - genetics
/ CRISPR-Associated Proteins - metabolism
/ Domains
/ Humanities and Social Sciences
/ RNA
/ RNA Processing, Post-Transcriptional
/ RNA, Guide, CRISPR-Cas Systems - genetics
/ Science
/ Spacer
