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...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2020-06-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/55143 |
| _version_ | 1811236204300992512 |
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| author | Joshua C Cofsky Deepti Karandur Carolyn J Huang Isaac P Witte John Kuriyan Jennifer A Doudna |
| author_facet | Joshua C Cofsky Deepti Karandur Carolyn J Huang Isaac P Witte John Kuriyan Jennifer A Doudna |
| author_sort | Joshua C Cofsky |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T12:04:47Z |
| format | Article |
| id | doaj.art-9de3aaaa86684a8983b6d4b9b0b19f07 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T12:04:47Z |
| publishDate | 2020-06-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-9de3aaaa86684a8983b6d4b9b0b19f072022-12-22T03:33:45ZengeLife Sciences Publications LtdeLife2050-084X2020-06-01910.7554/eLife.55143CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaksJoshua C Cofsky0https://orcid.org/0000-0001-5403-8555Deepti Karandur1Carolyn J Huang2Isaac P Witte3https://orcid.org/0000-0002-3879-0306John Kuriyan4https://orcid.org/0000-0002-4414-5477Jennifer A Doudna5https://orcid.org/0000-0001-9161-999XDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United StatesDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, United States; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United StatesDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United StatesDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United StatesDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, United States; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States; Department of Chemistry, University of California, Berkeley, Berkeley, United States; MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, United StatesDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, United States; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States; Department of Chemistry, University of California, Berkeley, Berkeley, United States; MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, United States; Innovative Genomics Institute, University of California, Berkeley, Berkeley, United States; Gladstone Institutes, University of California, San Francisco, San Francisco, United StatesType 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.https://elifesciences.org/articles/55143CRISPRgenome editingRNAdeoxyribonucleaseR-loop |
| spellingShingle | Joshua C Cofsky Deepti Karandur Carolyn J Huang Isaac P Witte John Kuriyan Jennifer A Doudna CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks eLife CRISPR genome editing RNA deoxyribonuclease R-loop |
| title | CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks |
| title_full | CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks |
| title_fullStr | CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks |
| title_full_unstemmed | CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks |
| title_short | CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks |
| title_sort | crispr cas12a exploits r loop asymmetry to form double strand breaks |
| topic | CRISPR genome editing RNA deoxyribonuclease R-loop |
| url | https://elifesciences.org/articles/55143 |
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