{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T14:45:09Z","timestamp":1785422709242,"version":"3.56.0"},"posted":{"date-parts":[[2025,1,5]]},"group-title":"Biochemistry","reference-count":31,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,1,5]],"date-time":"2025-01-05T00:00:00Z","timestamp":1736035200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["R35GM138080"],"award-info":[{"award-number":["R35GM138080"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["101158249"],"award-info":[{"award-number":["101158249"]}],"id":[{"id":"https:\/\/ror.org\/0472cxd90","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Helmholtz Institute for RNA-based Infection Research"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,30]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>CRISPR-Cas systems often rely on collateral cleavage of nucleic-acid substrates to combat recognized mobile genetic elements. Of the CRISPR-associated (Cas) RNA-guided effector nucleases, Cas12a2 stands out as the only known example exhibiting rapid collateral cleavage of three distinct substrates: single-stranded (ss)RNA, ssDNA, and double-stranded (ds)DNA, after activating upon binding cognate RNA. However, little is known about the underlying mechanisms of collateral cleavage. Here, we show, using enzyme kinetics and inhibition assays, that Cas12a2 preferentially cleaves collateral DNA over RNA substrates, even when RNA substrates are more abundant. Additionally, using enzyme mutants, enzyme kinetics, and plasmid cleavage assays, we determine that the dsDNA cleavage mechanism relies on the \u2018aromatic clamp\u2019 residues that stabilize unwound and distorted dsDNA in the RuvC nuclease active site. Leveraging the cleavage preference for collateral DNA, we demonstrate that RNA-activated Cas12a2 can readily cleave a ssDNA probe in the presence of high concentrations of non-target RNA, while an RNA-targeting Cas13a cannot. This work provides foundational kinetic and biochemical insights into the collateral cleavage mechanism and substrate preferences of Cas12a2, with immediate implications for understanding Cas12a2-based immunity and developing Cas12a2-based technologies.<\/jats:p>","DOI":"10.1101\/2025.01.05.631166","type":"posted-content","created":{"date-parts":[[2025,1,8]],"date-time":"2025-01-08T07:55:12Z","timestamp":1736322912000},"source":"Crossref","is-referenced-by-count":2,"title":["Target RNA-triggered CRISPR-Cas12a2 Preferentially Cleaves Collateral DNA over RNA"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-3248-8680","authenticated-orcid":false,"given":"Sobita","family":"Kunwar","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biochemistry, Utah State University, Logan, UT, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6285-8753","authenticated-orcid":false,"given":"Thomson","family":"Hallmark","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, Utah State University, Logan, UT, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3129-1819","authenticated-orcid":false,"given":"Sudeshna","family":"Manna","sequence":"additional","affiliation":[{"name":"Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), W\u00fcrzburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dylan","family":"Keiser","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, Utah State University, Logan, UT, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bronson","family":"Naegle","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, Utah State University, Logan, UT, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aaron","family":"Thomas","sequence":"additional","affiliation":[{"name":"College of Agriculture and Applied Sciences, Utah State University, Logan, UT, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0650-9943","authenticated-orcid":false,"given":"Chase L.","family":"Beisel","sequence":"additional","affiliation":[{"name":"Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), W\u00fcrzburg, Germany"},{"name":"Medical Faculty, University of W\u00fcrzburg, W\u00fcrzburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7403-3794","authenticated-orcid":false,"given":"Ryan N.","family":"Jackson","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, Utah State University, Logan, UT, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026060301450852000_2025.01.05.631166v2.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-022-05559-3"},{"key":"2026060301450852000_2025.01.05.631166v2.2","doi-asserted-by":"crossref","first-page":"582","DOI":"10.1038\/s41586-022-05560-w","article-title":"RNA targeting unleashes indiscriminate nuclease activity of CRISPR\u2013Cas12a2","volume":"613","year":"2023","journal-title":"Nature"},{"key":"2026060301450852000_2025.01.05.631166v2.3","doi-asserted-by":"publisher","DOI":"10.1126\/science.aaf5573"},{"key":"2026060301450852000_2025.01.05.631166v2.4","doi-asserted-by":"publisher","DOI":"10.1126\/science.aar6245"},{"key":"2026060301450852000_2025.01.05.631166v2.5","doi-asserted-by":"publisher","DOI":"10.1126\/science.aav7271"},{"key":"2026060301450852000_2025.01.05.631166v2.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41422-018-0022-x"},{"key":"2026060301450852000_2025.01.05.631166v2.7","doi-asserted-by":"publisher","DOI":"10.1101\/600890"},{"key":"2026060301450852000_2025.01.05.631166v2.8","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.0c00140"},{"key":"2026060301450852000_2025.01.05.631166v2.9","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.1c00204"},{"key":"2026060301450852000_2025.01.05.631166v2.10","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad715"},{"key":"2026060301450852000_2025.01.05.631166v2.11","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.2c01670"},{"key":"2026060301450852000_2025.01.05.631166v2.12","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202213920"},{"key":"2026060301450852000_2025.01.05.631166v2.13","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA120.012933"},{"key":"2026060301450852000_2025.01.05.631166v2.14","doi-asserted-by":"crossref","first-page":"138996","DOI":"10.1016\/j.ijbiomac.2024.138996","article-title":"CRISPR-Cas12a2-based rapid and sensitive detection system for target nucleic acid","volume":"290","year":"2025","journal-title":"Int. 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