CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks

Elife. 2020 Jun 10:9:e55143. doi: 10.7554/eLife.55143.

Abstract

Type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one strand of a double-helical DNA substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we detect intrinsic instability in DNA flanking the RNA-3' side of R-loops, which Cas12a can exploit to expose second-strand DNA for cutting. Interestingly, DNA flanking the RNA-5' side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

Keywords: CRISPR; E. coli; R-loop; RNA; biochemistry; chemical biology; deoxyribonuclease; genome editing.

Publication types

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

MeSH terms

  • Bacterial Proteins* / genetics
  • Bacterial Proteins* / metabolism
  • CRISPR-Associated Proteins* / genetics
  • CRISPR-Associated Proteins* / metabolism
  • CRISPR-Cas Systems / genetics*
  • DNA / genetics
  • DNA / metabolism
  • DNA Breaks, Double-Stranded*
  • Endodeoxyribonucleases* / genetics
  • Endodeoxyribonucleases* / metabolism
  • Escherichia coli / genetics
  • Gene Editing / methods*
  • R-Loop Structures / genetics*
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • RNA, Guide, CRISPR-Cas Systems / metabolism

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • RNA, Guide, CRISPR-Cas Systems
  • DNA
  • Cas12a protein
  • Endodeoxyribonucleases