A scoutRNA Is Required for Some Type V CRISPR-Cas Systems

Mol Cell. 2020 Aug 6;79(3):416-424.e5. doi: 10.1016/j.molcel.2020.06.022. Epub 2020 Jul 8.

Abstract

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.

Keywords: CRISPR-cas; Candidate Phyla Radiation (CPR) bacteria; Cas12c (C2c3); Cas12d (CasY); RuvC nuclease domain; crRNA; scoutRNA; tracrRNA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacteria / classification
  • Bacteria / genetics*
  • Bacteria / immunology
  • Bacteria / metabolism
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Base Sequence
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • DNA, Bacterial / metabolism
  • Endodeoxyribonucleases / genetics*
  • Endodeoxyribonucleases / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / immunology
  • Escherichia coli / metabolism
  • Genome, Bacterial / immunology*
  • Nucleic Acid Conformation
  • Phylogeny
  • RNA, Bacterial / chemistry
  • RNA, Bacterial / genetics*
  • RNA, Bacterial / metabolism
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • RNA, Small Untranslated / chemistry
  • RNA, Small Untranslated / genetics*
  • RNA, Small Untranslated / metabolism
  • Sequence Alignment
  • Sequence Homology, Nucleic Acid

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • RNA, Bacterial
  • RNA, Guide
  • RNA, Small Untranslated
  • Endodeoxyribonucleases