Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates
Abstract The sequence specificity and programmability of DNA binding and cleavage have enabled widespread applications of CRISPR-Cas12a in genetic engineering. As an RNA-guided CRISPR endonuclease, Cas12a engages a 20-base pair (bp) DNA segment by forming a three-stranded R-loop structure in which t...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-03-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-57703-y |
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| author | Kevin D. P. Aris Joshua C. Cofsky Honglue Shi Noor Al-Sayyad Ivan E. Ivanov Ashwin Balaji Jennifer A. Doudna Zev Bryant |
| author_facet | Kevin D. P. Aris Joshua C. Cofsky Honglue Shi Noor Al-Sayyad Ivan E. Ivanov Ashwin Balaji Jennifer A. Doudna Zev Bryant |
| author_sort | Kevin D. P. Aris |
| collection | DOAJ |
| description | Abstract The sequence specificity and programmability of DNA binding and cleavage have enabled widespread applications of CRISPR-Cas12a in genetic engineering. As an RNA-guided CRISPR endonuclease, Cas12a engages a 20-base pair (bp) DNA segment by forming a three-stranded R-loop structure in which the guide RNA hybridizes to the DNA target. Here we use single-molecule torque spectroscopy to investigate the dynamics and mechanics of R-loop formation of two widely used Cas12a orthologs at base-pair resolution. We directly observe kinetic intermediates corresponding to a ~5 bp initial RNA-DNA hybridization and a ~17 bp intermediate preceding R-loop completion, followed by transient DNA unwinding that extends beyond the 20 bp R-loop. The complex multistate landscape of R-loop formation is ortholog-dependent and shaped by target sequence, mismatches, and DNA supercoiling. A four-state kinetic model captures essential features of Cas12a R-loop dynamics and provides a biophysical framework for understanding Cas12a activity and specificity. |
| format | Article |
| id | doaj-art-bcbc1dc16ce444c29f5a04d08c8a12a0 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-bcbc1dc16ce444c29f5a04d08c8a12a02025-08-20T03:53:32ZengNature PortfolioNature Communications2041-17232025-03-0116111310.1038/s41467-025-57703-yDynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediatesKevin D. P. Aris0Joshua C. Cofsky1Honglue Shi2Noor Al-Sayyad3Ivan E. Ivanov4Ashwin Balaji5Jennifer A. Doudna6Zev Bryant7Biophysics Program, Stanford UniversityDepartment of Molecular and Cell Biology, University of California, BerkeleyHoward Hughes Medical Institute, University of California, BerkeleyDepartment of Bioengineering, Stanford UniversityDepartment of Bioengineering, Stanford UniversityBiophysics Program, Stanford UniversityDepartment of Molecular and Cell Biology, University of California, BerkeleyDepartment of Bioengineering, Stanford UniversityAbstract The sequence specificity and programmability of DNA binding and cleavage have enabled widespread applications of CRISPR-Cas12a in genetic engineering. As an RNA-guided CRISPR endonuclease, Cas12a engages a 20-base pair (bp) DNA segment by forming a three-stranded R-loop structure in which the guide RNA hybridizes to the DNA target. Here we use single-molecule torque spectroscopy to investigate the dynamics and mechanics of R-loop formation of two widely used Cas12a orthologs at base-pair resolution. We directly observe kinetic intermediates corresponding to a ~5 bp initial RNA-DNA hybridization and a ~17 bp intermediate preceding R-loop completion, followed by transient DNA unwinding that extends beyond the 20 bp R-loop. The complex multistate landscape of R-loop formation is ortholog-dependent and shaped by target sequence, mismatches, and DNA supercoiling. A four-state kinetic model captures essential features of Cas12a R-loop dynamics and provides a biophysical framework for understanding Cas12a activity and specificity.https://doi.org/10.1038/s41467-025-57703-y |
| spellingShingle | Kevin D. P. Aris Joshua C. Cofsky Honglue Shi Noor Al-Sayyad Ivan E. Ivanov Ashwin Balaji Jennifer A. Doudna Zev Bryant Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates Nature Communications |
| title | Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates |
| title_full | Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates |
| title_fullStr | Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates |
| title_full_unstemmed | Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates |
| title_short | Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates |
| title_sort | dynamic basis of supercoiling dependent dna interrogation by cas12a via r loop intermediates |
| url | https://doi.org/10.1038/s41467-025-57703-y |
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