CoChat
Sign Up
Cited by 14CRISPR and Genetic EngineeringRNA regulation and diseaseBacterial Genetics and Biotechnology

The DOI matches its Crossref record; 2 of the 2 references it cites were found in a registry or an index.

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 10.1093/nar/gkaf988PMC PMC12507515

Published version of a preprint

Open this paper in CoChat — free accountOpen PDF

Abstract

CRISPR-Cas12a mediates RNA-guided cleavage of double-stranded DNA in cis, after which it remains catalytically active and non-specifically cleaves single-stranded DNA 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-characterized 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 Escherichia 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. In total, our in vitro, in vivo, and in silico survey of Cas12a orthologues highlights key properties that drive their function in biotechnology applications.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1093/nar/gkaf988

Cites

2 of 2 references verified0 citation errors · 0 not checkable

Statistics

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Flagged first, then by citations

Research works printed but not linked: 70

  1. Barrangou, 2007, CRISPR provides acquired resistance against viruses in prokaryotes, Science, 315, 1709, 10.1126/science.1138140 · DOI · Paper page
  2. Makarova, 2015, An updated evolutionary classification of CRISPR–Cas systems, Nat Rev Microbiol, 13, 722, 10.1038/nrmicro3569 · DOI · Paper page
  3. Fonfara, 2016, The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA, Nature, 532, 517, 10.1038/nature17945 · DOI · Paper page
  4. Swarts, 2017, Structural basis for guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a, Mol Cell, 66, 221, 10.1016/j.molcel.2017.03.016 · DOI
  5. Zetsche, 2015, Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas System, Cell, 163, 759, 10.1016/j.cell.2015.09.038 · DOI · Paper page
  6. Kleinstiver, 2019, Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing, Nat Biotechnol, 37, 276, 10.1038/s41587-018-0011-0 · DOI
  7. Kim, 2016, Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells, Nat Biotechnol, 34, 863, 10.1038/nbt.3609 · DOI · Paper page
  8. Li, 2018, CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA, Cell Res, 28, 491, 10.1038/s41422-018-0022-x · DOI
  9. Chen, 2018, CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity, Science, 360, 436, 10.1126/science.aar6245 · DOI · Paper page
  10. Fuchs, Cas12a trans-cleavage can be modulated in vitro and is active on ssDNA, dsDNA, and RNA, 10.1101/600890 · DOI
  11. Kaminski, 2021, CRISPR-based diagnostics, Nat Biomed Eng, 5, 643, 10.1038/s41551-021-00760-7 · DOI
  12. Yang, 2023, Recent progress in aptamer and CRISPR-Cas12a based systems for non-nucleic target detection, Crit Rev Anal Chem, 0, 1
  13. Swarts, 2019, Mechanistic insights into the cis- and trans-acting DNase activities of Cas12a, Mol Cell, 73, 589, 10.1016/j.molcel.2018.11.021 · DOI
  14. Stella, 2018, Conformational activation promotes CRISPR-Cas12a catalysis and resetting of the endonuclease activity, Cell, 175, 1856, 10.1016/j.cell.2018.10.045 · DOI
  15. Yamano, 2016, Crystal structure of Cpf1 in complex with guide RNA and target DNA, Cell, 165, 949, 10.1016/j.cell.2016.04.003 · DOI
  16. Yamano, 2017, Structural basis for the canonical and non-canonical PAM recognition by CRISPR-Cpf1, Mol Cell, 67, 633, 10.1016/j.molcel.2017.06.035 · DOI · Paper page
  17. Dong, 2016, The crystal structure of Cpf1 in complex with CRISPR RNA, Nature, 532, 522, 10.1038/nature17944 · DOI · Paper page
  18. Strohkendl, 2024, Cas12a domain flexibility guides R-loop formation and forces RuvC resetting, Mol Cell, 84, 2717, 10.1016/j.molcel.2024.06.007 · DOI
  19. Singh, 2018, Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a), Proc Natl Acad Sci USA, 115, 5444, 10.1073/pnas.1718686115 · DOI
  20. Strohkendl, 2018, Kinetic basis for DNA target specificity of CRISPR-Cas12a, Mol Cell, 71, 816, 10.1016/j.molcel.2018.06.043 · DOI
  21. Wörle, 2022, Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain, Nucleic Acids Res, 50, 10153, 10.1093/nar/gkac767 · DOI · Paper page
  22. Wörle, 2021, Decoupling the bridge helix of Cas12a results in a reduced trimming activity, increased mismatch sensitivity and impaired conformational transitions, Nucleic Acids Res, 49, 5278, 10.1093/nar/gkab286 · DOI
  23. Parameshwaran, 2021, The bridge helix of Cas12a imparts selectivity in cis-DNA cleavage and regulates trans-DNA cleavage, FEBS Lett, 595, 892, 10.1002/1873-3468.14051 · DOI
  24. Saha, 2024, An alpha-helical lid guides the target DNA toward catalysis in CRISPR-Cas12a, Nat Commun, 15, 1473, 10.1038/s41467-024-45762-6 · DOI
  25. Cofsky, 2020, CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks, eLife, 9, e55143, 10.7554/eLife.55143 · DOI
  26. Naqvi, 2022, CRISPR-Cas12a-mediated DNA clamping triggers target-strand cleavage, Nat Chem Biol, 18, 1014, 10.1038/s41589-022-01082-8 · DOI
  27. van Aelst, 2019, The effect of DNA topology on observed rates of R-loop formation and DNA strand cleavage by CRISPR Cas12a, Genes, 10, 169, 10.3390/genes10020169 · DOI
  28. Son, 2021, Mg2+-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage, Proc Natl Acad Sci USA, 118, e2113747118, 10.1073/pnas.2113747118 · DOI
  29. Saha, 2020, Molecular dynamics reveals a DNA-induced dynamic switch triggering activation of CRISPR-Cas12a, J Chem Inf Model, 60, 6427, 10.1021/acs.jcim.0c00929 · DOI
  30. Zhang, 2019, Conformational dynamics and cleavage sites of Cas12a are modulated by complementarity between crRNA and DNA, iScience, 19, 492, 10.1016/j.isci.2019.08.005 · DOI
  31. Jeon, 2018, Direct observation of DNA target searching and cleavage by CRISPR-Cas12a, Nat Commun, 9, 2777, 10.1038/s41467-018-05245-x · DOI · Paper page
  32. Lamothe, 2023, Novel CRISPR-associated gene-editing systems discovered in metagenomic samples enable efficient and specific genome engineering, CRISPR J, 6, 243, 10.1089/crispr.2022.0089 · DOI
  33. Fuchs, 2022, Characterization of Cme and Yme thermostable Cas12a orthologs, Commun Biol, 5, 325, 10.1038/s42003-022-03275-2 · DOI
  34. Pan, 2025, Kinetic analysis and engineering of thermostable Cas12a for nucleic acid detection, Nucleic Acids Res, 53, gkaf509, 10.1093/nar/gkaf509 · DOI
  35. Wilkins, 1999, Protein identification and analysis tools in the ExPASy server, Methods Mol Biol, 112, 531
  36. Rueden, 2017, ImageJ2: imageJ for the next generation of scientific image data, BMC Bioinformatics, 18, 529, 10.1186/s12859-017-1934-z · DOI
  37. Kolbeck, 2024, Supercoiling-dependent DNA binding: quantitative modeling and applications to bulk and single-molecule experiments, Nucleic Acids Res, 52, 59, 10.1093/nar/gkad1055 · DOI
  38. Marcoline, 2022, Berkeley Madonna Version 10—a simulation package for solving mathematical models, CPT Pharmacometrics Syst Pharmacol, 11, 290, 10.1002/psp4.12757 · DOI
  39. Mullally, 2020, 5′ modifications to CRISPR–Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage, Nucleic Acids Res, 48, 6811, 10.1093/nar/gkaa477 · DOI
  40. Humphrey, 1996, VMD: visual molecular dynamics, J Mol Graph, 14, 33, 10.1016/0263-7855(96)00018-5 · DOI · Paper page
  41. Russell, 1992, Multiple protein sequence alignment from tertiary structure comparison: assignment of global and residue confidence levels, Proteins, 14, 309, 10.1002/prot.340140216 · DOI
  42. Varadi, 2022, AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models, Nucleic Acids Res, 50, D439, 10.1093/nar/gkab1061 · DOI · Paper page
  43. Kleinstiver, 2016, Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells, Nat Biotechnol, 34, 869, 10.1038/nbt.3620 · DOI · Paper page
  44. Tu, 2017, A ‘new lease of life’: fnCpf1 possesses DNA cleavage activity for genome editing in human cells, Nucleic Acids Res, 45, 11295, 10.1093/nar/gkx783 · DOI
  45. Gao, 2017, Engineered Cpf1 variants with altered PAM specificities, Nat Biotechnol, 35, 789, 10.1038/nbt.3900 · DOI · Paper page
  46. Schindele, 2020, Engineering CRISPR/LbCas12a for highly efficient, temperature-tolerant plant gene editing, Plant Biotechnol J, 18, 1118, 10.1111/pbi.13275 · DOI
  47. Clement, 2019, Accurate and rapid analysis of genome editing data from nucleases and base editors with CRISPResso2, Nat Biotechnol, 37, 224, 10.1038/s41587-019-0032-3 · DOI
  48. Schwede, 2003, SWISS-MODEL: an automated protein homology-modeling server, Nucleic Acids Res, 31, 3381, 10.1093/nar/gkg520 · DOI
  49. Tian, 2020, ff19SB: amino-acid-specific protein backbone parameters trained against quantum mechanics energy surfaces in solution, J Chem Theory Comput, 16, 528, 10.1021/acs.jctc.9b00591 · DOI
  50. Ivani, 2016, Parmbsc1: a refined force field for DNA simulations, Nat Methods, 13, 55, 10.1038/nmeth.3658 · DOI
  51. Banáš, 2010, Performance of molecular mechanics force fields for RNA simulations: stability of UUCG and GNRA hairpins, J Chem Theory Comput, 6, 3836, 10.1021/ct100481h · DOI
  52. Zgarbová, 2011, Refinement of the Cornell et al. nucleic acids force field based on reference quantum chemical calculations of glycosidic torsion profiles, J Chem Theory Comput, 7, 2886, 10.1021/ct200162x · DOI
  53. Jorgensen, 1983, Comparison of simple potential functions for simulating liquid water, J Chem Phys, 79, 926, 10.1063/1.445869 · DOI
  54. Li, 2013, Rational design of particle mesh Ewald compatible Lennard-Jones parameters for +2 metal cations in explicit solvent, J Chem Theory Comput, 9, 2733, 10.1021/ct400146w · DOI
  55. Nierzwicki, 2022, Principles of target DNA cleavage and the role of Mg2+ in the catalysis of CRISPR–Cas9, Nat Catal, 5, 912, 10.1038/s41929-022-00848-6 · DOI
  56. Ryckaert, 1977, Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes, J Comput Phys, 23, 327, 10.1016/0021-9991(77)90098-5 · DOI
  57. Turq, 1977, Brownian dynamics: its application to ionic solutions, J Chem Phys, 66, 3039, 10.1063/1.434317 · DOI
  58. Berendsen, 1984, Molecular dynamics with coupling to an external bath, J Chem Phys, 81, 3684, 10.1063/1.448118 · DOI
  59. Waskom, 2021, seaborn: statistical data visualization, J Open Source Softw, 6, 3021, 10.21105/joss.03021 · DOI · Paper page
  60. Connors, 1990, Chemical Kinetics: the Study of Reaction Rates in Solution
  61. Mohanraju, 2021, Development of a Cas12a-based genome editing tool for moderate thermophiles, The CRISPR J, 4, 82, 10.1089/crispr.2020.0086 · DOI
  62. Oppenheim, 1981, Separation of closed circular DNA from linear DNA by electrophoresis in two dimensions in agarose gels, Nucl Acids Res, 9, 6805, 10.1093/nar/9.24.6805 · DOI
  63. Naqvi, A gate and clamp regulate sequential DNA strand cleavage by CRISPR-Cas12a, 10.1101/2021.06.18.448962 · DOI
  64. Hwang, 2025, Enhanced trans-cleavage activity using CRISPR-Cas12a variant designed to reduce steric inhibition by cis-cleavage products, Biosens Bioelectron, 267, 116859, 10.1016/j.bios.2024.116859 · DOI
  65. Nguyen, 2020, Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection, Nat Commun, 11, 4906, 10.1038/s41467-020-18615-1 · DOI
  66. Sun, 2023, Nonspecific interactions between Cas12a and dsDNA located downstream of the PAM mediate target search and assist AsCas12a for DNA cleavage, Chem Sci, 14, 3839, 10.1039/D2SC05463A · DOI
  67. Soczek, 2025, CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation, Nucleic Acids Res, 53, gkae1192, 10.1093/nar/gkae1192 · DOI
  68. Murugan, 2020, CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects, J Biol Chem, 295, 5538, 10.1074/jbc.RA120.012933 · DOI
  69. Schelling, 2023, CRISPR-Cas effector specificity and cleavage site determine phage escape outcomes, PLoS Biol, 21, e3002065, 10.1371/journal.pbio.3002065 · DOI
  70. Fu, 2019, Target-dependent nickase activities of the CRISPR–Cas nucleases Cpf1 and Cas9, Nat Microbiol, 4, 888, 10.1038/s41564-019-0382-0 · DOI

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 ,
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2025-09-23
Volume
53
Issue
18
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkaf988
PMC
PMC12507515
OpenAlex
W4414969377
Cited by
14
References
2
Citations by year
2026: 13 · 2025: 1
FWCI
4.21
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 Health and Medical Research Council
  • The Gordon and Gretel Bootes
  • National Computing Infrastructure
  • NCRIS
  • Australian Government Research Training Program
  • National Institutes of Health (R01GM141329)
  • National Science Foundation (CHE-2144823, 2138259, 2138286, 2138307, 2137603, 2138296)
  • Alfred P. Sloan Foundation (FG-2023-20431)
  • Camille and Henry Dreyfus Foundation (TC-24-063)
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

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review