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

Lucas B. Harrington, Enbo Ma, Janice S. Chen, Isaac P. Witte, Dov Gertz, David Paez-Espino, Basem Al-Shayeb, Nikos C. Kyrpides, David Burstein, Jillian F. Banfield, Jennifer A. Doudna

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)416-424.e5
JournalMolecular Cell
Volume79
Issue number3
DOIs
StatePublished - 6 Aug 2020

Keywords

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

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