Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a WYL-Domain-Containing Accessory Protein

Mol Cell. 2018 Apr 19;70(2):327-339.e5. doi: 10.1016/j.molcel.2018.02.028. Epub 2018 Mar 15.

Abstract

Bacterial class 2 CRISPR-Cas systems utilize a single RNA-guided protein effector to mitigate viral infection. We aggregated genomic data from multiple sources and constructed an expanded database of predicted class 2 CRISPR-Cas systems. A search for novel RNA-targeting systems identified subtype VI-D, encoding dual HEPN domain-containing Cas13d effectors and putative WYL-domain-containing accessory proteins (WYL1 and WYL-b1 through WYL-b5). The median size of Cas13d proteins is 190 to 300 aa smaller than that of Cas13a-Cas13c. Despite their small size, Cas13d orthologs from Eubacterium siraeum (Es) and Ruminococcus sp. (Rsp) are active in both CRISPR RNA processing and targeting, as well as collateral RNA cleavage, with no target-flanking sequence requirements. The RspWYL1 protein stimulates RNA cleavage by both EsCas13d and RspCas13d, demonstrating a common regulatory mechanism for divergent Cas13d orthologs. The small size, minimal targeting constraints, and modular regulation of Cas13d effectors further expands the CRISPR toolkit for RNA manipulation and detection.

Keywords: CRISPR; CRISPR-Cas; RNA editing; RNA targeting; gene editing; genome engineering; type VI CRISPR-Cas systems.

Publication types

  • Research Support, N.I.H., Intramural

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*
  • Databases, Genetic
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Eubacterium / enzymology
  • Eubacterium / genetics
  • Gene Editing / methods*
  • Gene Expression Regulation, Bacterial
  • Nucleic Acid Conformation
  • Protein Domains
  • Protein Structure, Secondary
  • RNA Processing, Post-Transcriptional
  • RNA, Bacterial / chemistry
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism*
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • Ruminococcus / enzymology
  • Ruminococcus / genetics
  • Structure-Activity Relationship

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • RNA, Bacterial
  • RNA, Guide