{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T22:30:19Z","timestamp":1792794619456,"version":"4.5.0"},"reference-count":31,"publisher":"Oxford University Press (OUP)","issue":"11","license":[{"start":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T00:00:00Z","timestamp":1781568000000},"content-version":"vor","delay-in-days":8,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["R35GM138080"],"award-info":[{"award-number":["R35GM138080"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"name":"European Research Council Proof-of-Concept award","award":["101158249"],"award-info":[{"award-number":["101158249"]}],"id":[{"id":"https:\/\/ror.org\/0472cxd90","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Helmholtz Center for Infection Research (HZI) Singh-Chhatwal-Postdoctoral Fellowship","id":[{"id":"https:\/\/ror.org\/03d0p2685","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,6,8]]},"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":"10.1093\/nar\/gkag578","type":"journal-article","created":{"date-parts":[[2026,5,28]],"date-time":"2026-05-28T11:45:30Z","timestamp":1779968730000},"source":"Crossref","is-referenced-by-count":3,"title":["Target RNA-triggered CRISPR-Cas12a2 preferentially cleaves collateral DNA over RNA"],"prefix":"10.1093","volume":"54","author":[{"given":"Sobita","family":"Kunwar","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biochemistry, Utah State University , Logan, UT 84322 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomson","family":"Hallmark","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, Utah State University , Logan, UT 84322 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sudeshna","family":"Manna","sequence":"additional","affiliation":[{"name":"Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI) , W\u00fcrzburg 97080 ,","place":["Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dylan","family":"Keiser","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, Utah State University , Logan, UT 84322 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bronson","family":"Naegle","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, Utah State University , Logan, UT 84322 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aaron","family":"Thomas","sequence":"additional","affiliation":[{"name":"Center for Integrated Biosystems, Utah State University , Logan, UT 84322 ,","place":["United States"]}],"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 97080 ,","place":["Germany"]},{"name":"Medical Faculty, University of W\u00fcrzburg , W\u00fcrzburg 97080 ,","place":["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 84322 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,6,16]]},"reference":[{"key":"2026061611095746600_B1","doi-asserted-by":"publisher","first-page":"588","DOI":"10.1038\/s41586-022-05559-3","article-title":"Cas12a2 elicits abortive infection through RNA-triggered destruction of dsDNA","volume":"613","author":"Dmytrenko","year":"2023","journal-title":"Nature"},{"key":"2026061611095746600_B2","doi-asserted-by":"publisher","first-page":"582","DOI":"10.1038\/s41586-022-05560-w","article-title":"RNA targeting unleashes indiscriminate nuclease activity of CRISPR\u2013Cas12a2","volume":"613","author":"Bravo","year":"2023","journal-title":"Nature"},{"key":"2026061611095746600_B3","doi-asserted-by":"publisher","first-page":"aaf5573","DOI":"10.1126\/science.aaf5573","article-title":"C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector","volume":"353","author":"Abudayyeh","year":"2016","journal-title":"Science"},{"key":"2026061611095746600_B4","doi-asserted-by":"publisher","first-page":"436","DOI":"10.1126\/science.aar6245","article-title":"CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity","volume":"360","author":"Chen","year":"2018","journal-title":"Science"},{"key":"2026061611095746600_B5","doi-asserted-by":"publisher","first-page":"88","DOI":"10.1126\/science.aav7271","article-title":"Functionally diverse type V CRISPR-Cas systems","volume":"363","author":"Yan","year":"2019","journal-title":"Science"},{"key":"2026061611095746600_B6","doi-asserted-by":"publisher","first-page":"491","DOI":"10.1038\/s41422-018-0022-x","article-title":"CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA","volume":"28","author":"Li","year":"2018","journal-title":"Cell Res"},{"key":"2026061611095746600_B7","doi-asserted-by":"publisher","DOI":"10.1101\/600890","article-title":"Cas12a trans-cleavage can be modulated in vitro and is active on ssDNA, dsDNA, and RNA","author":"Fuchs","year":"2019"},{"key":"2026061611095746600_B8","doi-asserted-by":"publisher","first-page":"1474","DOI":"10.1021\/acs.biochem.0c00140","article-title":"Probing CRISPR-Cas12a nuclease activity using double-stranded DNA-templated fluorescent substrates","volume":"59","author":"Smith","year":"2020","journal-title":"Biochemistry"},{"key":"2026061611095746600_B9","doi-asserted-by":"publisher","first-page":"1785","DOI":"10.1021\/acssynbio.1c00204","article-title":"Recognition of DNA target formulations by CRISPR-Cas12a using a dsDNA reporter","volume":"10","author":"Smith","year":"2021","journal-title":"ACS Synth Biol"},{"key":"2026061611095746600_B10","doi-asserted-by":"publisher","first-page":"9894","DOI":"10.1093\/nar\/gkad715","article-title":"Unidirectional trans-cleaving behavior of CRISPR-Cas12a unlocks for an ultrasensitive assay using hybrid DNA reporters containing a 3\u2032 toehold","volume":"51","author":"Mohammad","year":"2023","journal-title":"Nucleic Acids Res"},{"key":"2026061611095746600_B11","doi-asserted-by":"publisher","first-page":"9826","DOI":"10.1021\/acs.analchem.2c01670","article-title":"Enzyme kinetics and detector sensitivity determine limits of detection of amplification-free CRISPR-Cas12 and CRISPR-Cas13 diagnostics","volume":"94","author":"Huyke","year":"2022","journal-title":"Anal Chem"},{"key":"2026061611095746600_B12","doi-asserted-by":"publisher","first-page":"e202213920","DOI":"10.1002\/anie.202213920","article-title":"A sensitive and nonoptical CRISPR detection mechanism by sizing double-stranded \u03bb DNA reporter","volume":"61","author":"Mohammad","year":"2022","journal-title":"Angew Chem Int Ed"},{"key":"2026061611095746600_B13","doi-asserted-by":"publisher","first-page":"5538","DOI":"10.1074\/jbc.RA120.012933","article-title":"CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects","volume":"295","author":"Murugan","year":"2020","journal-title":"J Biol Chem"},{"key":"2026061611095746600_B14","doi-asserted-by":"publisher","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","author":"Yu","year":"2025","journal-title":"Int J Biol Macromol"},{"key":"2026061611095746600_B15","doi-asserted-by":"publisher","first-page":"6414","DOI":"10.1093\/nar\/gkac462","article-title":"CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity","volume":"50","author":"Marino","year":"2022","journal-title":"Nucleic Acids Res"},{"key":"2026061611095746600_B16","doi-asserted-by":"publisher","first-page":"gkaf1293","DOI":"10.1093\/nar\/gkaf1293","article-title":"CRISPR\/Cas12a2 enables ultra-sensitive amplification-free RNA detection","volume":"53","author":"Jang","year":"2025","journal-title":"Nucleic Acids Res"},{"key":"2026061611095746600_B17","doi-asserted-by":"publisher","first-page":"152","DOI":"10.1038\/s41586-021-03222-x","article-title":"The kinetic landscape of an RNA-binding protein in cells","volume":"591","author":"Sharma","year":"2021","journal-title":"Nature"},{"key":"2026061611095746600_B18","doi-asserted-by":"publisher","first-page":"1188","DOI":"10.1101\/gr.849004","article-title":"WebLogo: a sequence logo generator","volume":"14","author":"Crooks","year":"2004","journal-title":"Genome Res"},{"key":"2026061611095746600_B19","doi-asserted-by":"publisher","first-page":"1025","DOI":"10.1016\/j.celrep.2018.06.105","article-title":"RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a","volume":"24","author":"Tambe","year":"2018","journal-title":"Cell Rep"},{"key":"2026061611095746600_B20","doi-asserted-by":"publisher","first-page":"247","DOI":"10.1016\/j.tibtech.2007.03.010","article-title":"Catalytic efficiency and kcat\/KM: a useful comparator?","volume":"25","author":"Eisenthal","year":"2007","journal-title":"Trends Biotechnol"},{"key":"2026061611095746600_B21","doi-asserted-by":"publisher","first-page":"70","DOI":"10.1016\/j.bbapap.2015.08.011","article-title":"Measuring specificity in multi-substrate\/product systems as a tool to investigate selectivity in vivo","volume":"1864","author":"Kuo","year":"2016","journal-title":"Biochim Biophys Acta (BBA) - Proteins and Proteomics"},{"key":"2026061611095746600_B22","doi-asserted-by":"publisher","first-page":"113382","DOI":"10.1016\/j.bios.2021.113382","article-title":"Detection of rare variant alleles using the AsCas12a double-stranded DNA trans-cleavage activity","volume":"189","author":"Lee\u00a0Yu","year":"2021","journal-title":"Biosens Bioelectron"},{"key":"2026061611095746600_B23","doi-asserted-by":"publisher","first-page":"1499","DOI":"10.1042\/BST20190564","article-title":"Making the cut(s): how Cas12a cleaves target and non-target DNA","volume":"47","author":"Swarts","year":"2019","journal-title":"Biochem Soc Trans"},{"key":"2026061611095746600_B24","doi-asserted-by":"publisher","first-page":"8651","DOI":"10.1039\/c2sm26089a","article-title":"Quantifying supercoiling-induced denaturation bubbles in DNA","volume":"8","author":"Adamcik","year":"2012","journal-title":"Soft Matter"},{"key":"2026061611095746600_B25","doi-asserted-by":"publisher","first-page":"281","DOI":"10.1016\/0022-2836(91)90212-O","article-title":"Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity","volume":"222","author":"Cayley","year":"1991","journal-title":"J Mol Biol"},{"key":"2026061611095746600_B26","doi-asserted-by":"publisher","first-page":"373","DOI":"10.1016\/j.molcel.2017.04.008","article-title":"RNA targeting by functionally orthogonal type VI-A CRISPR-Cas enzymes","volume":"66","author":"East-Seletsky","year":"2017","journal-title":"Mol Cell"},{"key":"2026061611095746600_B27","doi-asserted-by":"publisher","first-page":"4381","DOI":"10.1002\/j.1460-2075.1991.tb05016.x","article-title":"Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure","volume":"10","author":"Iwasaki","year":"1991","journal-title":"EMBO J"},{"key":"2026061611095746600_B28","doi-asserted-by":"publisher","first-page":"12217","DOI":"10.1073\/pnas.93.22.12217","article-title":"Resolution of Holliday junctions in genetic recombination: ruvC protein nicks DNA at the point of strand exchange","volume":"93","author":"Bennett","year":"1996","journal-title":"Proc Natl Acad Sci USA"},{"key":"2026061611095746600_B29","doi-asserted-by":"crossref","first-page":"4102","DOI":"10.1038\/s41467-019-11900-8","article-title":"RuvC uses dynamic probing of the Holliday junction to achieve sequence specificity and efficient resolution","volume":"10","author":"G\u00f3recka","year":"2019","journal-title":"Nat Commun"},{"key":"2026061611095746600_B30","doi-asserted-by":"publisher","first-page":"102996","DOI":"10.1016\/j.isci.2021.102996","article-title":"Kinetic analysis of Cas12a and Cas13a RNA-Guided nucleases for development of improved CRISPR-Based diagnostics","volume":"24","author":"Nalefski","year":"2021","journal-title":"iScience"},{"key":"2026061611095746600_B31","doi-asserted-by":"publisher","first-page":"2717","DOI":"10.1016\/j.molcel.2024.06.007e6","article-title":"Cas12a domain flexibility guides R-loop formation and forces RuvC resetting","volume":"84","author":"Strohkendl","year":"2024","journal-title":"Mol Cell"}],"container-title":["Nucleic Acids Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/54\/11\/gkag578\/68533164\/gkag578.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/54\/11\/gkag578\/68533164\/gkag578.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T15:10:04Z","timestamp":1781622604000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/nar\/article\/doi\/10.1093\/nar\/gkag578\/8708756"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,8]]},"references-count":31,"journal-issue":{"issue":"11","published-print":{"date-parts":[[2026,6,8]]}},"URL":"https:\/\/doi.org\/10.1093\/nar\/gkag578","relation":{},"ISSN":["0305-1048","1362-4962"],"issn-type":[{"value":"0305-1048","type":"print"},{"value":"1362-4962","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2026,6,24]]},"published":{"date-parts":[[2026,6,8]]},"article-number":"gkag578"}}