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

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...

Full description

Saved in:
Bibliographic Details
Published in:eLife 2020-06, Vol.9
Main Authors: Cofsky, Joshua C, Karandur, Deepti, Huang, Carolyn J, Witte, Isaac P, Kuriyan, John, Doudna, Jennifer A
Format: Article
Language:English
Subjects:
Architecture
Asymmetry
Bacterial genetics
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Biochemistry
Biochemistry and Chemical Biology
CRISPR
CRISPR-Associated Proteins - genetics
CRISPR-Associated Proteins - metabolism
CRISPR-Cas Systems - genetics
Crystal structure
Deoxyribonuclease
Deoxyribonucleic acid
DNA
DNA - genetics
DNA - metabolism
DNA Breaks, Double-Stranded
DNA damage
Double-stranded RNA
Endodeoxyribonucleases - genetics
Endodeoxyribonucleases - metabolism
Enzymes
Escherichia coli - genetics
Gene Editing - methods
Genetic research
Genome editing
Genomes
Genomics
Proteins
R-loop
R-Loop Structures - genetics
R-loops
Ribonucleic acid
RNA
RNA, Guide, CRISPR-Cas Systems - genetics
RNA, Guide, CRISPR-Cas Systems - metabolism
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:2050-084X
2050-084X
DOI:10.7554/eLife.55143