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

Nat Commun. 2019 Jun 11;10(1):2544. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • CRISPR-Associated Proteins / chemistry
  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Associated Proteins / metabolism
  • CRISPR-Cas Systems
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Nucleic Acid Conformation
  • Protein Domains
  • RNA Processing, Post-Transcriptional
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Ribonucleases / chemistry
  • Ribonucleases / genetics
  • Ribonucleases / metabolism*
  • Ruminococcus / enzymology
  • Ruminococcus / genetics*

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • RNA, Bacterial
  • RNA, Guide, CRISPR-Cas Systems
  • Ribonucleases