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

Bo Zhang,Siqi Wang,Jinying Lin,Yu Li,Jing Chen,Weiwei Ye,Han Jiang,Songying Ouyang,Yu-San Han,Qi Chen,Yangmiao Ye,Yiyang Chen,Vanja Perčulija

doi:10.1038/s41467-019-10507-3

Abstract

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 Mg2+ 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.

Highlights

Cas13d, the type VI-D clustered regularly interspaced short palindromic repeats (CRISPR)-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner
RNA cleavage is mediated by two R-X4-H motifs, which are characteristic of higher eukaryotes and prokaryotes nucleotide (HEPN)-binding domains[22,23,24,25,26]
In order to understand the structural basis of pre-CRISPR RNA (crRNA) processing and mature crRNA recognition by Cas13d, we solved the crystal structure of the uncultured Ruminococcus sp

Summary

Introduction

Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. The pre-crRNA processing is executed by the HEPN-2 domain Both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are recognized by UrCas13d. Class 2 systems (including types II, V, and VI) deploy a single Cas effector protein to accomplish CRISPR RNA (crRNA) biogenesis and interference[4,6,8]. Due to their simplicity, class 2 systems have become powerful tools for genome editing and other applications in life sciences[9,10,11,12,13,14,15,16]. We try to answer these questions by using high-resolution crystal structures of Cas13d and biochemical studies, and provide additional molecular information that can be used in rational design of the CRISPR-Cas13d system for a wide range of potential applications

Methods

Results

Conclusion