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PreprintCited by 14CRISPR and Genetic EngineeringRNA regulation and diseaseBacterial Genetics and Biotechnology

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CRISPR-Cas12a REC2 – NUC interactions drive target-strand cleavage and constrain trans cleavage

Open access
Newman, Anthony, Saha, Aakash, Starrs, Lora, Arantes, Pablo R., Palermo, Giulia, Burgio, Gaetan·2025·Nucleic Acids Research
DOI 10.1101/2025.03.23.644851PMC PMC12507515

Preprint — published as 10.1093/nar/gkaf988 · Paper page

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Abstract

CRISPR-Cas12a effects RNA-guided cleavage of dsDNA in cis , after which it remains catalytically active and non-specifically cleaves ssDNA in trans . Native host-defence by Cas12a employs cis cleavage, which can be repurposed for the genome editing of other organisms, and trans cleavage can be used for in vitro DNA detection. Cas12a orthologues have high structural similarity and a conserved mechanism of DNA cleavage, yet highly different efficacies when applied for genome editing or DNA detection. By comparing three well characterised Cas12a orthologues (FnCas12a, LbCas12a, and AsCas12a), we sought to determine what drives their different cis and trans cleavage, and how this relates to their applied function. We integrated in vitro DNA cleavage kinetics with molecular dynamics simulations, plasmid interference in E. coli , and genome editing in human cell lines. We report large differences in cis cleavage kinetics between orthologues, which may be driven by dynamic REC2-NUC interactions. We generated and tested REC2 and NUC mutants, including a hitherto unstudied ‘NUC loop’, integrity of which is critical for the function of Cas12 orthologues. In total, our in vitro, in vivo, and in silico survey of Cas12a orthologues highlights key properties that drive their function in biotechnology applications. Graphical abstract:

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Metadata

Authors
Newman, A., Saha, A., Starrs, L., Arantes, P. R., Palermo, G., & Burgio, G.
Affiliations
  • Australian National University (AU)
  • University of California, Riverside (US)
  • The Shine-Dalgarno Centre for RNA Innovation, Division of Genome Sciences and Cancer, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, 2601, Australia
ORCID
Venue
Nucleic Acids Research
Publisher
openRxiv
Published
2025-03-25
Volume
53
Issue
18
ISSN
0305-1048 (ISSN-L)
DOI
10.1101/2025.03.23.644851
PMC
PMC12507515
OpenAlex
W4414969377
Cited by
14
References
1
Citations by year
2026: 13 · 2025: 1
FWCI
4.18
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by)
  • ANU Open Research (Australian National University) (submittedVersion)
Funders
  • National Science Foundation (2137603, MCB160059, 2138259, 2138296, 2138307, BIO230007, 2138286, 2144823)
  • Alfred P. Sloan Foundation (FG-2023-20431)
  • Camille and Henry Dreyfus Foundation (TC-24-063)
  • Australian Government
  • National Institutes of Health (2138307, 2138286, 2138296, 2137603, 2138259)
  • Medical Research Council
  • National Health and Medical Research Council
  • National Institute of General Medical Sciences (R01GM141329)
  • Office of Advanced Cyberinfrastructure (#2138286, #2138296, 2138259, #2138307, 2137603)
  • Division of Chemistry (2138296, 2138307, 2137603, 2138259, 2138286)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
CRISPR/Cas12a, trans-cleavage, cis-cleavage, LbCas12a, AsCas12a, DNA cleavage kinetics, genome editing, DNA detection, molecular dynamics simulation

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