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Title: 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 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.  more » « less
Award ID(s):
1817593
PAR ID:
10473061
Author(s) / Creator(s):
; ; ; ; ;
Publisher / Repository:
eLife
Date Published:
Journal Name:
eLife
Volume:
9
ISSN:
2050-084X
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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