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

doi:10.1016/j.molcel.2020.06.022

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.

Authors

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
² School of Molecular Cell Biology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 6997801, Israel.
³ Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
⁴ Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
⁵ Department of Earth and Planetary Science, University of California, Berkeley, Berkeley, CA 94720, USA; School of Molecular Cell Biology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 6997801, Israel.
⁶ Department of Earth and Planetary Science, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
⁷ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Molecular Biophysics & Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Gladstone Institutes, University of California, San Francisco, San Francisco, CA 94114, USA. Electronic address: doudna@berkeley.edu.

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, CRISPR-Cas Systems / genetics
RNA, Guide, CRISPR-Cas Systems / 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, CRISPR-Cas Systems
RNA, Small Untranslated
Endodeoxyribonucleases

Abstract

Publication types

MeSH terms

Substances

Grants and funding