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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\u2032 side of R-loops, which Cas12a can exploit to expose second-strand DNA for cutting. Interestingly, DNA flanking the RNA-5\u2032 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.<\/jats:p>","DOI":"10.7554\/elife.55143","type":"journal-article","created":{"date-parts":[[2020,6,8]],"date-time":"2020-06-08T13:00:18Z","timestamp":1591621218000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.55143","source":"Crossref","is-referenced-by-count":126,"title":["CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks"],"prefix":"10.7554","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5403-8555","authenticated-orcid":true,"given":"Joshua C","family":"Cofsky","sequence":"first","affiliation":[{"name":"Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United 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