Advances in Double-Stranded DNA Targeting Technologies

Abstract

Double-stranded DNA (dsDNA) serves as a fundamental repository of genetic information and plays a pivotal role in the diagnosis and therapeutic management of diseases. However, the inherent stability of the DNA double helix under physiological conditions presents a challenge in accessing internal bases. To address this, various molecular targeting technologies have been developed, offering high specificity while destabilizing the DNA structure. This review provides a comprehensive overview of current dsDNA targeting tools, such as hybridization probes, modified nucleic acid probes, zinc finger proteins (ZFPs), transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system, Argonaute proteins (Agos), and the lambda exonuclease-pDNA system (λ Exo-pDNA), and some cutting-edge molecular tools. It delves into the mechanisms behind these technologies. It highlights their applications in diverse areas, including in vitro detection, in situ imaging, gene editing, and their integration with artificial intelligence (AI)-driven tools. Additionally, the review compares these techniques, discusses future technological opportunities, and identifies challenges in integrating these tools into diagnostic and therapeutic practices. By providing a holistic view of these rapidly evolving technologies, this review aims to fill a gap in the current literature and explore the future potential of dsDNA targeting innovations.

Keywords

artificial Intelligence / double-stranded DNA / gene targeting / gene editing / in situ Imaging / in vitro detection

References

[1] J. D. Watson and F. H. C. Crick, “Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid,” Nature 171 (1953): 737–738, https://doi.org/10.1038/171737a0.

[2] S. Takahashi and N. Sugimoto, “Watson–Crick Versus Hoogsteen Base Pairs: Chemical Strategy to Encode and Express Genetic Information in Life,” Accounts of Chemical Research 54 (2021): 2110–2120, https://doi.org/10.1021/acs.accounts.0c00734.

[3] K. A. Wilson, J. L. Kellie, and S. D. Wetmore, “DNA–Protein π-Interactions in Nature: Abundance, Structure, Composition and Strength of Contacts Between Aromatic Amino Acids and DNA Nucleobases or Deoxyribose Sugar,” Nucleic Acids Research 42 (2014): 6726–6741, https://doi.org/10.1093/nar/gku269.

[4] J. Sponer, K. E. Riley, and P. Hobza, “Nature and Magnitude of Aromatic Stacking of Nucleic Acid Bases,” Physical Chemistry Chemical Physics 10 (2008): 2595, https://doi.org/10.1039/b719370j.

[5] L. Z. Yang and L. L. Chen, “CRISPRdelight Illuminates Non-Repetitive Genomic Loci in Living Cells,” Nature Methods 21 (2024): 1591.

[6] C. Nieuwland, T. A. Hamlin, C. Fonseca Guerra, et al., “B-DNA Structure and Stability: The Role of Nucleotide Composition and Order,” ChemistryOpen 11 (2022): e202100231, https://doi.org/10.1002/open.202100231.

[7] D. Miyoshi, H. Karimata, and N. Sugimoto, “Hydration Regulates the Thermodynamic Stability of DNA Structures Under Molecular Crowding Conditions,” Nucleosides Nucleotides & Nucleic Acids 26 (2007): 589–595, https://doi.org/10.1080/15257770701490282.

[8] P. L. Privalov and C. Crane-Robinson, “Role of Water in the Formation of Macromolecular Structures,” European Biophysics Journal 46 (2017): 203–224, https://doi.org/10.1007/s00249-016-1161-y.

[9] B. R. Voldborg, L. Damstrup, M. Spang-Thomsen, et al., “Epidermal Growth Factor Receptor (EGFR) and EGFR Mutations, Function and Possible Role in Clinical Trials,” Annals of Oncology 8 (1997): 1197–1206, https://doi.org/10.1023/A:1008209720526.

[10] C. R. Chong and P. A. Jänne, “The Quest to Overcome Resistance to EGFR-Targeted Therapies in Cancer,” Nature Medicine 19 (2013): 1389–1400, https://doi.org/10.1038/nm.3388.

[11] S. Rasheed Abubakir and H. J. Hidayat, “Molecular Study of Colorectal Carcinoma and EGFR Gene Mutations,” Cellular and Molecular Biology (Noisy-Le-Grand) 69 (2023): 177–182, https://doi.org/10.14715/cmb/2023.69.9.27.

[12] L. Huang, Z. Guo, F. Wang, et al., “KRAS Mutation: From Undruggable to Druggable in Cancer,” Signal Transduction and Targeted Therapy 6 (2021): 386, https://doi.org/10.1038/s41392-021-00780-4.

[13] M. Herreros-Villanueva, C. C. Chen, S. S. Yuan, et al., “KRAS Mutations: Analytical Considerations,” Clinica Chimica Acta 431 (2014): 211–220, https://doi.org/10.1016/j.cca.2014.01.049.

[14] H. Chen, M. Zhang, L. Bai, et al., “Coexistence of a Novel SV2B-ALK, EML4-ALK Double-Fusion in a Lung Poorly Differentiated Adenocarcinoma Patient and Response to Alectinib: A Case Report and Literature Review,” Frontiers in Oncology 14 (2024): 1453259, https://doi.org/10.3389/fonc.2024.1453259.

[15] H. Nie, S. Zhang, L. Wang, et al., “Synthesis of Novel Deuterated EGFR/ALK Dual-Target Inhibitors and Their Activity Against Non-Small Cell Lung Cancer,” European Journal of Medicinal Chemistry 283 (2025): 117146, https://doi.org/10.1016/j.ejmech.2024.117146.

[16] Q. E. Yang, X. Ma, M. Li, et al., “Evolution of Triclosan Resistance Modulates Bacterial Permissiveness to Multidrug Resistance Plasmids and Phages,” Nature Communications 15 (2024): 3654, https://doi.org/10.1038/s41467-024-48006-9.

[17] J. Moura De Sousa, R. Balbontín, P. Durão, et al., “Multidrug-Resistant Bacteria Compensate for the Epistasis Between Resistances,” PLoS Biology 15 (2017): e2001741, https://doi.org/10.1371/journal.pbio.2001741.

[18] R. Zheng, L. Zhang, R. Parvin, et al., “Progress and Perspective of CRISPR-Cas9 Technology in Translational Medicine,” Advanced Science 10 (2023): 2300195, https://doi.org/10.1002/advs.202300195.

[19] D. K. Sahel, L. K. Vora, A. Saraswat, et al., “CRISPR/Cas9 Genome Editing for Tissue-Specific In Vivo Targeting: Nanomaterials and Translational Perspective,” Advanced Science 10 (2023): 2207512, https://doi.org/10.1002/advs.202207512.

[20] L. Amoasii, J. C. W. Hildyard, H. Li, et al., “Gene Editing Restores Dystrophin Expression in a Canine Model of Duchenne Muscular Dystrophy,” Science 362 (2018): 86–91, https://doi.org/10.1126/science.aau1549.

[21] O. Santiago-Fernández, F. G. Osorio, V. Quesada, et al., “Development of a CRISPR/Cas9-Based Therapy for Hutchinson–Gilford Progeria Syndrome,” Nature Medicine 25 (2019): 423–426, https://doi.org/10.1038/s41591-018-0338-6.

[22] H. Frangoul, D. Altshuler, M. D. Cappellini, et al., “CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia,” New England Journal of Medicine 384 (2021): 252–260, https://doi.org/10.1056/NEJMoa2031054.

[23] A. Sharma, J. J. Boelens, M. Cancio, et al., “CRISPR-Cas9 Editing of the HBG1 and HBG2 Promoters to Treat Sickle Cell Disease,” New England Journal of Medicine 389 (2023): 820–832, https://doi.org/10.1056/NEJMoa2215643.

[24] D. B. Sulis, X. Jiang, C. Yang, et al., “Multiplex CRISPR Editing of Wood for Sustainable Fiber Production,” Science 381 (2023): 216–221, https://doi.org/10.1126/science.add4514.

[25] G. J. Knott and J. A. Doudna, “CRISPR-Cas Guides the Future of Genetic Engineering,” Science 361 (2018): 866–869, https://doi.org/10.1126/science.aat5011.

[26] P. Bhowmik, D. Konkin, P. Polowick, et al., “CRISPR/Cas9 Gene Editing in Legume Crops: Opportunities and Challenges,” Legume Science 3 (2021): e96, https://doi.org/10.1002/leg3.96.

[27] W. J. Lyzenga, C. J. Pozniak, and S. Kagale, “Advanced Domestication: Harnessing the Precision of Gene Editing in Crop Breeding,” Plant Biotechnology Journal 19 (2021): 660–670, https://doi.org/10.1111/pbi.13576.

[28] N. C. Seeman, “Nucleic Acid Junctions and Lattices,” Journal of Theoretical Biology 99 (1982): 237–247, https://doi.org/10.1016/0022-5193(82)90002-9.

[29] D. Yang, M. R. Hartman, T. L. Derrien, et al., “DNA Materials: Bridging Nanotechnology and Biotechnology,” Accounts of Chemical Research 47 (2014): 1902–1911, https://doi.org/10.1021/ar5001082.

[30] M. Madsen and K. V. Gothelf, “Chemistries for DNA Nanotechnology,” Chemical Reviews 119 (2019): 6384–6458, https://doi.org/10.1021/acs.chemrev.8b00570.

[31] L. Zhang, J. Chen, M. He, et al., “Molecular Dynamics Simulation-Guided Toehold Mediated Strand Displacement Probe for Single-Nucleotide Variants Detection,” Exploration 2 (2022): 20210265, https://doi.org/10.1002/EXP.20210265.

[32] F. C. Simmel, B. Yurke, and H. R. Singh, “Principles and Applications of Nucleic Acid Strand Displacement Reactions,” Chemical Reviews 119 (2019): 6326–6369, https://doi.org/10.1021/acs.chemrev.8b00580.

[33] J. Wu, J. Lv, X. Zheng, et al., “Hybridization Chain Reaction and its Applications in Biosensing,” Talanta 234 (2021): 122637, https://doi.org/10.1016/j.talanta.2021.122637.

[34] S. Bi, S. Yue, and S. Zhang, “Hybridization Chain Reaction: A Versatile Molecular Tool for Biosensing, Bioimaging, and Biomedicine,” Chemical Society Reviews 46 (2017): 4281–4298, https://doi.org/10.1039/C7CS00055C.

[35] L. Zhao, Y. Song, and H. Xu, “Catalytic Hairpin Self-Assembly for Biosensing: Classification, Influencing Factors, and Applications,” TrAC Trends in Analytical Chemistry 171 (2024): 117508, https://doi.org/10.1016/j.trac.2023.117508.

[36] M. Wang, Y. Tang, Y. Chen, et al., “Catalytic Hairpin Assembly-Programmed Formation of Clickable Nucleic Acids for Electrochemical Detection of Liver Cancer Related Short Gene,” Analytica Chimica Acta 1045 (2019): 77–84, https://doi.org/10.1016/j.aca.2018.08.057.

[37] X. Wang and A. Son, “Effects of Pretreatment on the Denaturation and Fragmentation of Genomic DNA for DNA Hybridization,” Environmental Science: Processes & Impacts 15 (2013): 2204, https://doi.org/10.1039/c3em00457k.

[38] D. S. S. Marpaung, A. O. Y. Sinaga, D. Damayanti, et al., “Bridging Biological Samples to Functional Nucleic Acid Biosensor Applications: Current Enzymatic-Based Strategies for Single-Stranded DNA Generation,” Analytical Sciences 40 (2024): 1225–1237, https://doi.org/10.1007/s44211-024-00566-y.

[39] J. G. Gall and M. L. Pardue, “Formation and Detection of RNA-DNA Hybrid Molecules in Cytological Preparations,” Proceedings of the National Academy of Sciences of the United States of America 63 (1969): 378–383, https://doi.org/10.1073/pnas.63.2.378.

[40] H. M. Padilla-Nash, L. Barenboim-Stapleton, M. J. Difilippantonio, et al., “Spectral Karyotyping Analysis of Human and Mouse Chromosomes,” Nature Protocols 1 (2006): 3129–3142, https://doi.org/10.1038/nprot.2006.358.

[41] G. T. Rudkin and B. D. Stollar, “High Resolution Detection of DNA–RNA Hybrids In Situ by Indirect Immunofluorescence,” Nature 265 (1977): 472–473, https://doi.org/10.1038/265472a0.

[42] M. R. Speicher, S. G. Ballard, and D. C. Ward, “Karyotyping Human Chromosomes by Combinatorial Multi-Fluor FISH,” Nature Genetics 12 (1996): 368–375, https://doi.org/10.1038/ng0496-368.

[43] M. J. Rust, M. Bates, and X. Zhuang, “Sub-Diffraction-Limit Imaging by Stochastic Optical Reconstruction Microscopy (STORM),” Nature Methods 3 (2006): 793–796, https://doi.org/10.1038/nmeth929.

[44] S. W. Hell and J. Wichmann, “Breaking the Diffraction Resolution Limit by Stimulated Emission: Stimulated-Emission-Depletion Fluorescence Microscopy,” Optics Letters 19 (1994): 780, https://doi.org/10.1364/OL.19.000780.

[45] E. Betzig, G. H. Patterson, R. Sougrat, et al., “Imaging Intracellular Fluorescent Proteins at Nanometer Resolution,” Science 313 (2006): 1642–1645, https://doi.org/10.1126/science.1127344.

[46] M. G. L. Gustafsson, “Surpassing the Lateral Resolution Limit by a Factor of Two Using Structured Illumination Microscopy,” Journal of Microscopy 198 (2000): 82–87, https://doi.org/10.1046/j.1365-2818.2000.00710.x.

[47] F. Chen, P. W. Tillberg, and E. S. Boyden, “Expansion Microscopy,” Science 347 (2015): 543–548, https://doi.org/10.1126/science.1260088.

[48] J. Chen, D. Mcswiggen, and E. Ünal, “Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis,” Journal of Visualized Experiments 135 (2018): e57774.

[49] J. L. Nielsen, “Assigning Function to Phylogeny: MAR-FISH,” Methods in Molecular Biology 2246 (2021): 225–236.

[50] J. J. N. Perkel, “Starfish Enterprise: Finding RNA Patterns in Single Cells,” Nature 572 (2019): 549–551, https://doi.org/10.1038/d41586-019-02477-9.

[51] K. H. Chen, A. N. Boettiger, J. R. Moffitt, et al., “Spatially Resolved, Highly Multiplexed RNA Profiling in Single Cells,” Science 348 (2015): aaa6090, https://doi.org/10.1126/science.aaa6090.

[52] M. Zhang, S. W. Eichhorn, B. Zingg, et al., “Spatially Resolved Cell Atlas of the Mouse Primary Motor Cortex by MERFISH,” Nature 598 (2021): 137–143, https://doi.org/10.1038/s41586-021-03705-x.

[53] T. Lu, C. E. Ang, and X. Zhuang, “Spatially Resolved Epigenomic Profiling of Single Cells in Complex Tissues,” Cell 185 (2022): 4448–4464.e17, https://doi.org/10.1016/j.cell.2022.09.035.

[54] J. Das, I. Ivanov, E. H. Sargent, et al., “DNA Clutch Probes for Circulating Tumor DNA Analysis,” Journal of the American Chemical Society 138 (2016): 11009–11016, https://doi.org/10.1021/jacs.6b05679.

[55] G. A. Wang, X. Xie, H. Mansour, et al., “Expanding Detection Windows for Discriminating Single Nucleotide Variants Using Rationally Designed DNA Equalizer Probes,” Nature Communications 11 (2020): 5473, https://doi.org/10.1038/s41467-020-19269-9.

[56] D. Huang, H. Deng, J. Zhou, et al., “Mismatch-Guided Deoxyribonucleic Acid Assembly Enables Ultrasensitive and Multiplex Detection of Low-Allele-Fraction Variants in Clinical Samples,” Journal of the American Chemical Society 145 (2023): 20412–20421, https://doi.org/10.1021/jacs.3c05879.

[57] J. Lee, I. S. Park, E. Jung, et al., “Direct, Sequence-Specific Detection of dsDNA Based on Peptide Nucleic Acid and Graphene Oxide Without Requiring Denaturation,” Biosensors & Bioelectronics 62 (2014): 140–144, https://doi.org/10.1016/j.bios.2014.06.028.

[58] M. Lyu, L. Kong, Z. Yang, et al., “PNA-Assisted DNAzymes to Cleave Double-Stranded DNA for Genetic Engineering With High Sequence Fidelity,” Journal of the American Chemical Society 143 (2021): 9724–9728, https://doi.org/10.1021/jacs.1c03129.

[59] M. E. Everly, R. G. Emehiser, and P. J. Hrdlicka, “Recognition of Mixed-Sequence Double-Stranded DNA Regions Using Chimeric Invader/LNA Probes,” Organic & Biomolecular Chemistry 23 (2024): 619–628.

[60] T. Huang, Y. Han, Y. Chen, et al., “RLP System: A Single-Tube Two-Step Approach With Dual Amplification Cascades for Rapid Identification of EGFR T790M,” Analytica Chimica Acta 1287 (2024): 342126, https://doi.org/10.1016/j.aca.2023.342126.

[61] P. E. Nielsen, M. Egholm, R. H. Berg, et al., “Sequence-Selective Recognition of DNA by Strand Displacement With a Thymine-Substituted Polyamide,” Science 254 (1991): 1497–1500, https://doi.org/10.1126/science.1962210.

[62] C. Li, G. Zhang, S. Mohapatra, et al., “Machine Learning Guides Peptide Nucleic Acid Flow Synthesis and Sequence Design,” Advanced Science 9 (2022): 2201988, https://doi.org/10.1002/advs.202201988.

[63] P. Paulasova and F. Pellestor, “The Peptide Nucleic Acids (PNAs): A New Generation of Probes for Genetic and Cytogenetic Analyses,” Annales de Genetique 47 (2004): 349–358, https://doi.org/10.1016/j.anngen.2004.07.001.

[64] F. Pellestor and P. Paulasova, “The Peptide Nucleic Acids (PNAs), Powerful Tools for Molecular Genetics and Cytogenetics,” European Journal of Human Genetics 12 (2004): 694–700, https://doi.org/10.1038/sj.ejhg.5201226.

[65] I. V. Smolina, V. V. Demidov, V. A. Soldatenkov, et al., “End Invasion of Peptide Nucleic Acids (PNAs) With Mixed-Base Composition Into Linear DNA Duplexes,” Nucleic Acids Research 33 (2005): e146, https://doi.org/10.1093/nar/gni151.

[66] E. S. Baker, J. W. Hong, B. S. Gaylord, et al., “PNA/dsDNA Complexes: Site Specific Binding and dsDNA Biosensor Applications,” Journal of the American Chemical Society 128 (2006): 8484–8492, https://doi.org/10.1021/ja060069s.

[67] M. S. Hejazi, M. H. Pournaghi-Azar, and E. Alipour, “Development of a Novel Electrochemical Biosensor for Detection and Discrimination of DNA Sequence and Single Base Mutation in dsDNA Samples Based on PNA-dsDNA Hybridization—A New Platform Technology,” Electroanalysis 23 (2011): 503.

[68] X. Liang, M. Liu, and M. Komiyama, “Recognition of Target Site in Various Forms of DNA and RNA by Peptide Nucleic Acid (PNA): From Fundamentals to Practical Applications,” Bulletin of the Chemical Society of Japan 94 (2021): 1737–1756, https://doi.org/10.1246/bcsj.20210086.

[69] Y. Aiba, M. Shibata, and O. Shoji, “Sequence-Specific Recognition of Double-Stranded DNA by Peptide Nucleic Acid Forming Double-Duplex Invasion Complex,” Applied Sciences-Basel 12 (2022): 3677, https://doi.org/10.3390/app12073677.

[70] T. V. Pochechueva, N. Schwenzer, T. Kohl, et al., “3D Super-Resolution Nuclear Q-FISH Imaging Reveals Cell-Cycle-Related Telomere Changes,” International Journal of Molecular Sciences 25 (2024): 3183, https://doi.org/10.3390/ijms25063183.

[71] S. K. Singh, A. A. Koshkin, J. Wengel, et al., “LNA (Locked Nucleic Acids): Synthesis and High-Affinity Nucleic Acid Recognition,” Chemical Communications 4 (1998): 455–456, https://doi.org/10.1039/a708608c.

[72] S. P. Adhikari, P. Vukelich, D. C. Guenther, et al., “Recognition of Double-Stranded DNA Using LNA-Modified Toehold Invader Probes,” Organic & Biomolecular Chemistry 19 (2021): 9276–9290, https://doi.org/10.1039/D1OB01888D.

[73] S. P. Sau, T. S. Kumar, and P. J. Hrdlicka, “Invader LNA: Efficient Targeting of Short Double Stranded DNA,” Organic & Biomolecular Chemistry 8 (2010): 2028, https://doi.org/10.1039/b923465a.

[74] P. M. Moreno, S. Geny, Y. V. Pabon, et al., “Development of Bis-Locked Nucleic Acid (bisLNA) Oligonucleotides for Efficient Invasion of Supercoiled Duplex DNA,” Nucleic Acids Research 41 (2013): 3257–3273, https://doi.org/10.1093/nar/gkt007.

[75] D. Renneberg and C. J. Leumann, “Exploring Hoogsteen and Reversed-Hoogsteen Duplex and Triplex Formation With Tricyclo-DNA Purine Sequences,” Chembiochem 5 (2004): 1114–1118, https://doi.org/10.1002/cbic.200400069.

[76] V. Kumar, N. Brodyagin, and E. Rozners, “Triplex-Forming Peptide Nucleic Acids With Extended Backbones,” Chembiochem 21 (2020): 3410–3416, https://doi.org/10.1002/cbic.202000432.

[77] M. Alvira and R. Eritja, “Triplex-Stabilizing Properties of Parallel Clamps Carrying LNA Derivatives at the Hoogsteen Strand,” Chemistry & Biodiversity 7 (2010): 376–382, https://doi.org/10.1002/cbdv.200900230.

[78] G. Singh and V. Monga, “Peptide Nucleic Acids: Recent Developments in the Synthesis and Backbone Modifications,” Bioorganic Chemistry 141 (2023): 106860, https://doi.org/10.1016/j.bioorg.2023.106860.

[79] J. Miller, A. D. Mclachlan, and A. Klug, “Repetitive Zinc-Binding Domains in the Protein Transcription Factor IIIA From Xenopus Oocytes,” EMBO Journal 4 (1985): 1609–1614, https://doi.org/10.1002/j.1460-2075.1985.tb03825.x.

[80] M. Elrod-Erickson, M. A. Rould, L. Nekludova, et al., “Zif268 Protein-DNA Complex Refined at 1.6 A: A Model System for Understanding Zinc Finger-DNA Interactions,” Structure (London, England) 4 (1996): 1171–1180, https://doi.org/10.1016/S0969-2126(96)00125-6.

[81] D. Jantz, B. T. Amann, G. J. Gatto, et al., “The Design of Functional DNA-Binding Proteins Based on Zinc Finger Domains,” Chemical Reviews 104 (2004): 789–800, https://doi.org/10.1021/cr020603o.

[82] T. Gaj, J. Guo, Y. Kato, et al., “Targeted Gene Knockout by Direct Delivery of Zinc-Finger Nuclease Proteins,” Nature Methods 9 (2012): 805–807, https://doi.org/10.1038/nmeth.2030.

[83] Y. Santiago, E. Chan, P. Q. Liu, et al., “Targeted Gene Knockout in Mammalian Cells by Using Engineered Zinc-Finger Nucleases,” Proceedings of the National Academy of Sciences of the United States of America 105 (2008): 5809–5814, https://doi.org/10.1073/pnas.0800940105.

[84] J. Wang, G. Friedman, Y. Doyon, et al., “Targeted Gene Addition to a Predetermined Site in the Human Genome Using a ZFN-Based Nicking Enzyme,” Genome Research 22 (2012): 1316–1326, https://doi.org/10.1101/gr.122879.111.

[85] L. Mukhametzyanova, L. T. Schmitt, J. Torres-Rivera, et al., “Activation of Recombinases at Specific DNA Loci by Zinc-Finger Domain Insertions,” Nature Biotechnology 42 (2024): 1844–1854, https://doi.org/10.1038/s41587-023-02121-y.

[86] F. D. Urnov, J. C. Miller, Y.-L. Lee, et al., “Highly Efficient Endogenous Human Gene Correction Using Designed Zinc-Finger Nucleases,” Nature 435 (2005): 646–651, https://doi.org/10.1038/nature03556.

[87] F. D. Urnov, E. J. Rebar, M. C. Holmes, et al., “Genome Editing With Engineered Zinc Finger Nucleases,” Nature Reviews Genetics 11 (2010): 636–646, https://doi.org/10.1038/nrg2842.

[88] J. Abramson, J. Adler, J. Dunger, et al., “Accurate Structure Prediction of Biomolecular Interactions With AlphaFold3,” Nature 630 (2024): 493–500, https://doi.org/10.1038/s41586-024-07487-w.

[89] S. Katayama, M. Watanabe, Y. Kato, et al., “Engineering of Zinc Finger Nucleases Through Structural Modeling Improves Genome Editing Efficiency in Cells,” Advanced Science 11 (2024): 2310255, https://doi.org/10.1002/advs.202310255.

[90] C. S. Fang, K.-S. Kim, D. T. Ha, et al., “Washing-Free Electrochemical Detection of Amplified Double-Stranded DNAs Using a Zinc Finger Protein,” Analytical Chemistry 90 (2018): 4776–4782, https://doi.org/10.1021/acs.analchem.8b00143.

[91] M. S. Kim, G. Stybayeva, J. Y. Lee, et al., “A Zinc Finger Protein Array for the Visual Detection of Specific DNA Sequences for Diagnostic Applications,” Nucleic Acids Research 39 (2011): e29, https://doi.org/10.1093/nar/gkq1214.

[92] M. S. Kim and J. Kim, “Multiplexed Detection of Pathogen-Specific DNA Using Engineered Zinc Finger Proteins Without Target Amplification,” Analytical Methods 8 (2016): 6696–6700, https://doi.org/10.1039/C6AY02102F.

[93] S. Noh, D. T. Ha, H. Yang, et al., “Sensitive and Direct Electrochemical Detection of Double-Stranded DNA Utilizing Alkaline Phosphatase-Labelled Zinc Finger Proteins,” Analyst 140 (2015): 3947–3952, https://doi.org/10.1039/C5AN00623F.

[94] D. T. Ha, S. Ghosh, C. H. Ahn, et al., “Pathogen-Specific DNA Sensing With Engineered Zinc Finger Proteins Immobilized on a Polymer Chip,” Analyst 143 (2018): 4009–4016, https://doi.org/10.1039/C8AN00395E.

[95] D. Williams, L. M. Glasstetter, T. T. Jong, et al., “High-Throughput Screening for Small-Molecule Stabilizers of Misfolded Glucocerebrosidase in Gaucher Disease and Parkinson's Disease,” Proceedings of the National Academy of Sciences of the United States of America 121 (2024): e2406009121, https://doi.org/10.1073/pnas.2406009121.

[96] J. Forner, D. Kleinschmidt, E. H. Meyer, et al., “Targeted Knockout of a Conserved Plant Mitochondrial Gene by Genome Editing,” Nature Plants 9 (2023): 1818–1831, https://doi.org/10.1038/s41477-023-01538-2.

[97] J. Forner, D. Kleinschmidt, E. H. Meyer, et al., “Targeted Introduction of Heritable Point Mutations Into the Plant Mitochondrial Genome,” Nature Plants 8 (2022): 245–256, https://doi.org/10.1038/s41477-022-01108-y.

[98] N. Sun and H. Zhao, “Transcription Activator-Like Effector Nucleases (TALENs): A Highly Efficient and Versatile Tool for Genome Editing,” Biotechnology and Bioengineering 110 (2013): 1811–1821, https://doi.org/10.1002/bit.24890.

[99] J. K. Joung and J. D. Sander, “TALENs: A Widely Applicable Technology for Targeted Genome Editing,” Nature Reviews Molecular Cell Biology 14 (2013): 49–55, https://doi.org/10.1038/nrm3486.

[100] C. L. Ramirez, J. E. Foley, D. A. Wright, et al., “Unexpected Failure Rates for Modular Assembly of Engineered Zinc Fingers,” Nature Methods 5 (2008): 374–375, https://doi.org/10.1038/nmeth0508-374.

[101] Á. Muñoz-López, B. Buchmuller, J. Wolffgramm, et al., “Designer Receptors for Nucleotide-Resolution Analysis of Genomic 5-Methylcytosine by Cellular Imaging,” Angewandte Chemie International Edition in English 59 (2020): 8927.

[102] G. Kubik and D. Summerer, “Achieving Single-Nucleotide Resolution of 5-Methylcytosine Detection With TALEs,” Chembiochem 16 (2015): 228–231, https://doi.org/10.1002/cbic.201402408.

[103] Y. Miyanari, C. Ziegler-Birling, and M.-E. Torres-Padilla, “Live Visualization of Chromatin Dynamics With Fluorescent TALEs,” Nature Structural & Molecular Biology 20 (2013): 1321–1324, https://doi.org/10.1038/nsmb.2680.

[104] S.-S. Xie, X.-Y. Qiu, L.-Y. Zhu, et al., “Assembly of TALE-Based DNA Scaffold for the Enhancement of Exogenous Multi-Enzymatic Pathway,” Journal of Biotechnology 296 (2019): 69–74, https://doi.org/10.1016/j.jbiotec.2019.03.008.

[105] M. Jinek, K. Chylinski, I. Fonfara, et al., “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity,” Science 337 (2012): 816–821, https://doi.org/10.1126/science.1225829.

[106] M. M. Kaminski, O. O. Abudayyeh, J. S. Gootenberg, et al., “CRISPR-Based Diagnostics,” Nature Biomedical Engineering 5 (2021): 643–656, https://doi.org/10.1038/s41551-021-00760-7.

[107] W. L. Delano, “The PyMol Molecular Graphics System,” Proteins Structure Function and Bioinformatics 30 (2002): 442.

[108] R. Hajian, S. Balderston, T. Tran, et al., “Detection of Unamplified Target Genes via CRISPR-Cas9 Immobilized on a Graphene Field-Effect Transistor,” Nature Biomedical Engineering 3 (2019): 427–437, https://doi.org/10.1038/s41551-019-0371-x.

[109] S. Balderston, J. J. Taulbee, E. Celaya, et al., “Discrimination of Single-Point Mutations in Unamplified Genomic DNA via Cas9 Immobilized on a Graphene Field-Effect Transistor,” Nature Biomedical Engineering 5 (2021): 713–725, https://doi.org/10.1038/s41551-021-00706-z.

[110] B. Chen, L. A. Gilbert, B. A. Cimini, et al., “Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System,” Cell 155 (2013): 1479–1491, https://doi.org/10.1016/j.cell.2013.12.001.

[111] W. Deng, X. Shi, R. Tjian, et al., “CASFISH: CRISPR/Cas9-Mediated In Situ Labeling of Genomic Loci in Fixed Cells,” Proceedings of the National Academy of Sciences of the United States of America 112 (2015): 11870–11875, https://doi.org/10.1073/pnas.1515692112.

[112] H. Wang, M. Nakamura, T. R. Abbott, et al., “CRISPR-Mediated Live Imaging of Genome Editing and Transcription,” Science 365 (2019): 1301–1305, https://doi.org/10.1126/science.aax7852.

[113] X. Y. Lyu, Y. Deng, X. Y. Huang, et al., “CRISPR FISHer Enables High-Sensitivity Imaging of Nonrepetitive DNA in Living Cells Through Phase Separation-Mediated Signal Amplification,” Cell Research 32 (2022): 969–981, https://doi.org/10.1038/s41422-022-00712-z.

[114] Z. Zhang, X. Rong, T. Xie, et al., “Fluorogenic CRISPR for Genomic DNA Imaging,” Nature Communications 15 (2024): 934, https://doi.org/10.1038/s41467-024-45163-9.

[115] C. Dai, D. Kong, C. Chen, et al., “Graphene Transistors for In Vitro Detection of Health Biomarkers,” Advanced Functional Materials 33 (2023): 2301948, https://doi.org/10.1002/adfm.202301948.

[116] D. Kong, S. Zhang, X. Ma, et al., “DNA Logical Computing on a Transistor for Cancer Molecular Diagnosis,” Angewandte Chemie International Edition 63 (2024): e202407039, https://doi.org/10.1002/anie.202407039.

[117] A. Pannone, A. Raj, H. Ravichandran, et al., “Robust Chemical Analysis With Graphene Chemosensors and Machine Learning,” Nature 634 (2024): 572–578, https://doi.org/10.1038/s41586-024-08003-w.

[118] J. S. Chen, E. Ma, L. B. Harrington, et al., “CRISPR-Cas12a Target Binding Unleashes Indiscriminate Single-Stranded DNase Activity,” Science 360 (2018): 436–439, https://doi.org/10.1126/science.aar6245.

[119] S. Wang, S. Wang, Y. Tang, et al., “Detection of Klebsiella Pneumonia DNA and ESBL Positive Strains by PCR-Based CRISPR-LbCas12a System,” Frontiers in Microbiology 14 (2023): 1128261, https://doi.org/10.3389/fmicb.2023.1128261.

[120] S.-Y. Li, Q.-X. Cheng, J.-M. Wang, et al., “CRISPR-Cas12a-Assisted Nucleic Acid Detection,” Cell Discovery 4 (2018): 20, https://doi.org/10.1038/s41421-018-0028-z.

[121] S.-Y. Li, Q.-X. Cheng, J.-K. Liu, et al., “CRISPR-Cas12a has Both Cis- and Trans-Cleavage Activities on Single-Stranded DNA,” Cell Research 28 (2018): 491–493, https://doi.org/10.1038/s41422-018-0022-x.

[122] L. B. Harrington, D. Burstein, J. S. Chen, et al., “Programmed DNA Destruction by Miniature CRISPR-Cas14 Enzymes,” Science 362 (2018): 839–842, https://doi.org/10.1126/science.aav4294.

[123] X. Yin, L. Yang, X. Sun, et al., “Development and Validation of Sensitive and Rapid CRISPR/Cas12-Based PCR Method to Detect Hazelnut in Unlabeled Products,” Food Chemistry 438 (2024): 137952, https://doi.org/10.1016/j.foodchem.2023.137952.

[124] L. Li, S. Li, N. Wu, et al., “HOLMESv2: A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation,” ACS Synthetic Biology 8 (2019): 2228–2237, https://doi.org/10.1021/acssynbio.9b00209.

[125] A. A. Parikesit, R. Hermantara, K. Gregorius, et al., “Designing Hybrid CRISPR-Cas12 and LAMP Detection Systems for Treatment-Resistant Plasmodium Falciparum With In Silico Method,” Narra J 3 (2023): e301, https://doi.org/10.52225/narra.v3i3.301.

[126] A. Mahas, N. Hassan, R. Aman, et al., “LAMP-Coupled CRISPR-Cas12a Module for Rapid and Sensitive Detection of Plant DNA Viruses,” Viruses 13 (2021): 466, https://doi.org/10.3390/v13030466.

[127] A. Sen, M. Masetty, S. Weerakoon, et al., “Paper-Based Loop-Mediated Isothermal Amplification and CRISPR Integrated Platform for On-Site Nucleic Acid Testing of Pathogens,” Biosensors & Bioelectronics 257 (2024): 116292, https://doi.org/10.1016/j.bios.2024.116292.

[128] F. Zhao, Y. Hu, Z. Fan, et al., “Rapid and Sensitive One-Tube Detection of Mpox virus Using RPA-Coupled CRISPR-Cas12 Assay,” Cell Reports Methods 3 (2023): 100620, https://doi.org/10.1016/j.crmeth.2023.100620.

[129] B. Dai, A. Xiang, D. Qu, et al., “Rapid and Sensitive Assay of Helicobacter pylori With One-Tube RPA-CRISPR/Cas12 by Portable Array Detector for Visible Analysis of Thermostatic Nucleic Acid Amplification,” Frontiers in Microbiology 13 (2022): 858247, https://doi.org/10.3389/fmicb.2022.858247.

[130] M. J. Kellner, J. G. Koob, J. S. Gootenberg, et al., “SHERLOCK: Nucleic Acid Detection With CRISPR Nucleases,” Nature Protocols 14 (2019): 2986–3012, https://doi.org/10.1038/s41596-019-0210-2.

[131] W. Humphrey, A. Dalke, and K. Schulten, “VMD: Visual Molecular Dynamics,” Journal of Molecular Graphics 14 (1996): 33–38, https://doi.org/10.1016/0263-7855(96)00018-5.

[132] L. Z. Yang, Y. H. Min, Y. X. Liu, et al., “CRISPR-Array-Mediated Imaging of Non-Repetitive and Multiplex Genomic Loci in Living Cells,” Nature Methods 21 (2024): 1646–1657, https://doi.org/10.1038/s41592-024-02333-3.

[133] A. R. Eggers, K. Chen, K. M. Soczek, et al., “Rapid DNA Unwinding Accelerates Genome Editing by Engineered CRISPR-Cas9,” Cell 187 (2024): 3249–3261.e14, https://doi.org/10.1016/j.cell.2024.04.031.

[134] P. Mali, L. Yang, K. M. Esvelt, et al., “RNA-Guided Human Genome Engineering via Cas9,” Science 339 (2013): 823–826, https://doi.org/10.1126/science.1232033.

[135] A. C. Komor, Y. B. Kim, M. S. Packer, et al., “Programmable Editing of a Target Base in Genomic DNA Without Double-Stranded DNA Cleavage,” Nature 533 (2016): 420–424, https://doi.org/10.1038/nature17946.

[136] Y. B. Kim, A. C. Komor, J. M. Levy, et al., “Increasing the Genome-Targeting Scope and Precision of Base Editing With Engineered Cas9-Cytidine Deaminase Fusions,” Nature Biotechnology 35 (2017): 371–376, https://doi.org/10.1038/nbt.3803.

[137] A. C. Komor, K. T. Zhao, M. S. Packer, et al., “Improved Base Excision Repair Inhibition and Bacteriophage Mu Gam Protein Yields C:G-to-T:A Base Editors With Higher Efficiency and Product Purity,” Science Advances 3 (2017): eaao4774, https://doi.org/10.1126/sciadv.aao4774.

[138] L. W. Koblan, J. L. Doman, C. Wilson, et al., “Improving Cytidine and Adenine Base Editors by Expression Optimization and Ancestral Reconstruction,” Nature Biotechnology 36 (2018): 843–846, https://doi.org/10.1038/nbt.4172.

[139] N. M. Gaudelli, A. C. Komor, H. A. Rees, et al., “Programmable Base Editing of A•T to G•C in Genomic DNA Without DNA Cleavage,” Nature 551 (2017): 464–471, https://doi.org/10.1038/nature24644.

[140] J. L. Doman, A. Raguram, G. A. Newby, et al., “Evaluation and Minimization of Cas9-Independent Off-Target DNA Editing by Cytosine Base Editors,” Nature Biotechnology 38 (2020): 620–628, https://doi.org/10.1038/s41587-020-0414-6.

[141] A. V. Anzalone, P. B. Randolph, J. R. Davis, et al., “Search-and-Replace Genome Editing Without Double-Strand Breaks or Donor DNA,” Nature 576 (2019): 149–157, https://doi.org/10.1038/s41586-019-1711-4.

[142] M. Marzec, A. Braszewska-Zalewska, and G. Hensel, “Prime Editing: A New Way for Genome Editing,” Trends in Cell Biology 30 (2020): 257–259, https://doi.org/10.1016/j.tcb.2020.01.004.

[143] K. Chen, H. Han, S. Zhao, et al., “Lung and Liver Editing by Lipid Nanoparticle Delivery of a Stable CRISPR-Cas9 Ribonucleoprotein,” Nature Biotechnology 42 (2024): 1564–1574.

[144] W. Xing, D. Li, W. Wang, et al., “Conformational Dynamics of CasX (Cas12e) in Mediating DNA Cleavage Revealed by Single-Molecule FRET,” Nucleic Acids Research 52 (2024): 9014–9027, https://doi.org/10.1093/nar/gkae604.

[145] J.-J. Liu, N. Orlova, B. L. Oakes, et al., “Author Correction: CasX Enzymes Comprise a Distinct Family of RNA-Guided Genome Editors,” Nature 568 (2019): E8, https://doi.org/10.1038/s41586-019-1084-8.

[146] J.-J. Liu, N. Orlova, B. L. Oakes, et al., “CasX Enzymes Comprise a Distinct Family of RNA-Guided Genome Editors,” Nature 566 (2019): 218–223, https://doi.org/10.1038/s41586-019-0908-x.

[147] P. Pausch, K. M. Soczek, D. A. Herbst, et al., “DNA Interference States of the Hypercompact CRISPR–CasF Effector,” Nature Structural & Molecular Biology 28 (2021): 652–661, https://doi.org/10.1038/s41594-021-00632-3.

[148] C. Xin, J. Yin, S. Yuan, et al., “Comprehensive Assessment of Miniature CRISPR-Cas12f Nucleases for Gene Disruption,” Nature Communications 13 (2022): 5623, https://doi.org/10.1038/s41467-022-33346-1.

[149] A. Sun, C.-P. Li, Z. Chen, et al., “The Compact Casπ (Cas12l) ‘Bracelet’ Provides a Unique Structural Platform for DNA Manipulation,” Cell Research 33 (2023): 229–244, https://doi.org/10.1038/s41422-022-00771-2.

[150] W. Chen, J. Ma, Z. Wu, et al., “Cas12n Nucleases, Early Evolutionary Intermediates of type V CRISPR, Comprise a Distinct Family of Miniature Genome Editors,” Molecular Cell 83 (2023): 2768–2780.e6, https://doi.org/10.1016/j.molcel.2023.06.014.

[151] T. Yamano, B. Zetsche, R. Ishitani, et al., “Structural Basis for the Canonical and Non-Canonical PAM Recognition by CRISPR-Cpf1,” Molecular Cell 67 (2017): 633–645.e3, https://doi.org/10.1016/j.molcel.2017.06.035.

[152] S. Lu, X. Tong, Y. Han, et al., “Fast and Sensitive Detection of SARS-CoV-2 RNA Using Suboptimal Protospacer Adjacent Motifs for Cas12a,” Nature Biomedical Engineering 6 (2022): 286–297, https://doi.org/10.1038/s41551-022-00861-x.

[153] G. W. Goldberg, J. M. Spencer, D. O. Giganti, et al., “Engineered Dual Selection for Directed Evolution of SpCas9 PAM Specificity,” Nature Communications 12 (2021): 349, https://doi.org/10.1038/s41467-020-20650-x.

[154] I. M. Slaymaker, L. Gao, B. Zetsche, et al., “Rationally Engineered Cas9 Nucleases With Improved Specificity,” Science 351 (2016): 84–88, https://doi.org/10.1126/science.aad5227.

[155] J. S. Chen, Y. S. Dagdas, B. P. Kleinstiver, et al., “Enhanced Proofreading Governs CRISPR-Cas9 Targeting Accuracy,” Nature 550 (2017): 407–410, https://doi.org/10.1038/nature24268.

[156] B. P. Kleinstiver, V. Pattanayak, M. S. Prew, et al., “High-Fidelity CRISPR-Cas9 Nucleases With no Detectable Genome-Wide Off-Target Effects,” Nature 529 (2016): 490–495, https://doi.org/10.1038/nature16526.

[157] L. Zhang, W. He, R. Fu, et al., “Guide-Specific Loss of Efficiency and Off-Target Reduction With Cas9 Variants,” Nucleic Acids Research 51 (2023): 9880–9893, https://doi.org/10.1093/nar/gkad702.

[158] J. L. Schmid-Burgk, L. Gao, D. Li, et al., “Highly Parallel Profiling of Cas9 Variant Specificity,” Molecular Cell 78 (2020): 794–800.e8, https://doi.org/10.1016/j.molcel.2020.02.023.

[159] D. Matsumoto, E. Matsugi, K. Kishi, et al., “SpCas9-HF1 Enhances Accuracy of Cell Cycle-Dependent Genome Editing by Increasing HDR Efficiency, and by Reducing Off-Target Effects and Indel Rates,” Molecular Therapy Nucleic Acids 35 (2024): 102124, https://doi.org/10.1016/j.omtn.2024.102124.

[160] H. Manghwar, B. Li, X. Ding, et al., “CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off-Target Evaluation, and Strategies to Mitigate Off-Target Effects,” Advanced Science 7 (2020): 1902312, https://doi.org/10.1002/advs.201902312.

[161] J. A. Doudna, “The Promise and Challenge of Therapeutic Genome Editing,” Nature 578 (2020): 229–236, https://doi.org/10.1038/s41586-020-1978-5.

[162] J. P. K. Bravo, M.-S. Liu, G. N. Hibshman, et al., “Structural Basis for Mismatch Surveillance by CRISPR–Cas9,” Nature 603 (2022): 343–347, https://doi.org/10.1038/s41586-022-04470-1.

[163] J. G. Doench, N. Fusi, M. Sullender, et al., “Optimized sgRNA Design to Maximize Activity and Minimize Off-Target Effects of CRISPR-Cas9,” Nature Biotechnology 34 (2016): 184–191, https://doi.org/10.1038/nbt.3437.

[164] D. D. Kocak, E. A. Josephs, V. Bhandarkar, et al., “Increasing the Specificity of CRISPR Systems With Engineered RNA Secondary Structures,” Nature Biotechnology 37 (2019): 657–666, https://doi.org/10.1038/s41587-019-0095-1.

[165] Y. Dang, G. Jia, J. Choi, et al., “Optimizing sgRNA Structure to Improve CRISPR-Cas9 Knockout Efficiency,” Genome Biology 16 (2015): 280, https://doi.org/10.1186/s13059-015-0846-3.

[166] J. X. Chen, Y. T. Hu, C. C. Zhang, et al., “sgRNA Structure Optimization and PTG/Cas9 System Synergistically Boost Gene Knockout Efficiency in an Insect,” International Journal of Biological Macromolecules 280 (2024): 135413, https://doi.org/10.1016/j.ijbiomac.2024.135413.

[167] J. A. Ruffolo, S. Nayfach, J. Gallagher, et al., “Design of Highly Functional Genome Editors by Modeling the Universe of CRISPR-Cas Sequences,” preprint, bioRxiv, April 22, 2024, https://doi.org/10.1101/2024.04.22.590591.

[168] E. Nguyen, M. Poli, M. G. Durrant, et al., “Sequence Modeling and Design From Molecular to Genome Scale With Evo,” Science 386 (2024): eado9336, https://doi.org/10.1126/science.ado9336.

[169] S. Ryazansky, A. Kulbachinskiy, and A. A. Aravin, “The Expanded Universe of Prokaryotic Argonaute Proteins,” mBio 9, no. 6 (2018): e01935-18, https://doi.org/10.1128/mBio.01935-18.

[170] K. S. Makarova, Y. I. Wolf, J. Van Der Oost, et al., “Prokaryotic Homologs of Argonaute Proteins are Predicted to Function as Key Components of a Novel System of Defense Against Mobile Genetic Elements,” Biology Direct 4 (2009): 29, https://doi.org/10.1186/1745-6150-4-29.

[171] X. Gao, K. Shang, K. Zhu, et al., “Nucleic-Acid-Triggered NADase Activation of a Short Prokaryotic Argonaute,” Nature 625 (2024): 822–831, https://doi.org/10.1038/s41586-023-06665-6.

[172] L. Wang, W. Chen, C. Zhang, et al., “Molecular Mechanism for Target Recognition, Dimerization, and Activation of Pyrococcus furiosus Argonaute,” Molecular Cell 84 (2024): 675–686.e4, https://doi.org/10.1016/j.molcel.2024.01.004.

[173] Q. Liu, X. Guo, G. Xun, et al., “Argonaute Integrated Single-Tube PCR System Enables Supersensitive Detection of Rare Mutations,” Nucleic Acids Research 49 (2021): e75, https://doi.org/10.1093/nar/gkab274.

[174] H. Jang, J. Song, S. Kim, et al., “ANCA: Artificial Nucleic Acid Circuit With Argonaute Protein for One-Step Isothermal Detection of Antibiotic-Resistant Bacteria,” Nature Communications 14 (2023): 8033, https://doi.org/10.1038/s41467-023-43899-4.

[175] Y. Li, Y. Liu, Q. Zhang, et al., “An Argonaute-Mediated Bio-Barcode Bioassay for One-Tube and On-Site Detection of Staphylococcus Aureus,” Sensors and Actuators B: Chemical 410 (2024): 135713, https://doi.org/10.1016/j.snb.2024.135713.

[176] Y. Zhao, M. Yang, C. Zhou, et al., “Establishment of a Simple, Sensitive, and Specific ASFV Detection Method Based on Pyrococcus furiosus Argonaute,” Biosensors & Bioelectronics 254 (2024): 116230, https://doi.org/10.1016/j.bios.2024.116230.

[177] J. Kou, Y. Li, Z. Zhao, et al., “Simultaneous Dual-Gene Test of Methicillin-Resistant Staphylococcus Aureus Using an Argonaute-Centered Portable and Visual Biosensor,” Small 20 (2024): e2311764, https://doi.org/10.1002/smll.202311764.

[178] Z. Wu, L. Yu, W. Shi, et al., “Argonaute Protein-Based Nucleic Acid Detection Technology,” Frontiers in Microbiology 14 (2023): 1255716, https://doi.org/10.3389/fmicb.2023.1255716.

[179] B. A. Graver, N. Chakravarty, and K. V. Solomon, “Prokaryotic Argonautes for In Vivo Biotechnology and Molecular Diagnostics,” Trends in Biotechnology 42 (2024): 61–73, https://doi.org/10.1016/j.tibtech.2023.06.010.

[180] R. Vaiskunaite, J. Vainauskas, J. J. L. Morris, et al., “Programmable Cleavage of Linear Double-Stranded DNA by Combined Action of Argonaute CbAgo From Clostridium Butyricum and Nuclease Deficient RecBC Helicase From E. Coli,” Nucleic Acids Research 50 (2022): 4616–4629, https://doi.org/10.1093/nar/gkac229.

[181] S. Sun, D. Xu, L. Zhu, et al., “A Programmable, DNA-Exclusively-Guided Argonaute DNase and Its Higher Cleavage Specificity Achieved by 5'-Hydroxylated Guide,” Biomolecules 12 (2022): 1340, https://doi.org/10.3390/biom12101340.

[182] K. Z. Lee, M. A. Mechikoff, A. Kikla, et al., “NgAgo Possesses Guided DNA Nicking Activity,” Nucleic Acids Research 49 (2021): 9926–9937, https://doi.org/10.1093/nar/gkab757.

[183] K. Subramanian, W. Rutvisuttinunt, W. Scott, et al., “The Enzymatic Basis of Processivity in λ Exonuclease,” Nucleic Acids Research 31 (2003): 1585–1596, https://doi.org/10.1093/nar/gkg266.

[184] H. Yang, L. Zhang, X. Kang, et al., “Reaction Pathway Differentiation Enabled Fingerprinting Signal for Single Nucleotide Variant Detection,” Advanced Science 12, no. 12 (2025): 2412680.

[185] R. Kovall and B. W. Matthews, “Toroidal Structure of Lambda-Exonuclease,” Science 277 (1997): 1824–1827, https://doi.org/10.1126/science.277.5333.1824.

[186] S. Fu, J. Li, J. Chen, et al., “Bacteriophage λ Exonuclease and a 5′-Phosphorylated DNA Guide Allow PAM-Independent Targeting of Double-Stranded Nucleic Acids,” Nature Biotechnology 43, no. 7 (2025): 1144–1155.

[187] A. Johnson-Buck, X. Su, M. D. Giraldez, et al., “Kinetic Fingerprinting to Identify and Count Single Nucleic Acids,” Nature Biotechnology 33 (2015): 730–732, https://doi.org/10.1038/nbt.3246.

[188] X. Su, L. Li, S. Wang, et al., “Single-Molecule Counting of Point Mutations by Transient DNA Binding,” Scientific Reports 7 (2017): 43824, https://doi.org/10.1038/srep43824.

[189] J. Ha, H. Cho, T. G. Lee, et al., “Cytogenetic Testing by Fluorescence In Situ Hybridization is Improved by Plasma Cell Sorting in Multiple Myeloma,” Scientific Reports 12 (2022): 8287, https://doi.org/10.1038/s41598-022-11676-w.

[190] J. Jedrzkiewicz, D. Sirohi, N. Uvejzovic, et al., “RAI1 Alternate Probe Identifies Additional Gastroesophageal Adenocarcinoma Cases as Amplified Following Equivocal HER2 Fluorescence In Situ Hybridization Testing: Experience From a National Reference Laboratory,” Modern Pathology 35 (2022): 549–553, https://doi.org/10.1038/s41379-021-00933-4.

[191] D. C. Ferguson, A. M. Boroujeni, and T. Zheng, “ERBB2 Amplification Status in 67 Salivary Duct Carcinomas Assessed by Immunohistochemistry, Fluorescence In Situ Hybridization, and Targeted Exome Sequencing,” Modern Pathology 35 (2022): 895–902, https://doi.org/10.1038/s41379-021-00999-0.

[192] Z. Jian, T. Song, Z. Zhang, et al., “Deep Learning Method for Detecting Fluorescence Spots in Cancer Diagnostics via Fluorescence In Situ Hybridization,” Scientific Reports 14 (2024): 27231, https://doi.org/10.1038/s41598-024-78571-4.

[193] H. Chang, Y. Zhang, F. Yang, et al., “ctDNA Detection Based on DNA Clutch Probes and Strand Exchange Mechanism,” Frontiers in Chemistry 6 (2018): 530, https://doi.org/10.3389/fchem.2018.00530.

[194] F. Ahour, M. H. Pournaghi-Azar, E. Alipour, et al., “Detection and Discrimination of Recombinant Plasmid Encoding Hepatitis C virus core/E1 Gene Based on PNA and Double-Stranded DNA Hybridization,” Biosensors & Bioelectronics 45 (2013): 287–291, https://doi.org/10.1016/j.bios.2013.01.063.

[195] K. Abe, T. Kumagai, C. Takahashi, et al., “Detection of Pathogenic Bacteria by Using Zinc Finger Protein Fused With Firefly Luciferase,” Analytical Chemistry 84 (2012): 8028–8032, https://doi.org/10.1021/ac3018845.

[196] Y. Liu, X. Liu, D. Wei, et al., “CoHIT: A One-Pot Ultrasensitive ERA-CRISPR System for Detecting Multiple Same-Site Indels,” Nature Communications 15 (2024): 5014, https://doi.org/10.1038/s41467-024-49414-7.

[197] J. He, X. Hu, X. Weng, et al., “Efficient, Specific and Direct Detection of Double-Stranded DNA Targets Using Cas12f1 Nucleases and Engineered Guide RNAs,” Biosensors & Bioelectronics 260 (2024): 116428, https://doi.org/10.1016/j.bios.2024.116428.

[198] Y. Wang, H. Chen, K. Lin, et al., “Ultrasensitive Single-Step CRISPR Detection of Monkeypox Virus in Minutes With a Vest-Pocket Diagnostic Device,” Nature Communications 15 (2024): 3279, https://doi.org/10.1038/s41467-024-47518-8.

[199] C. Y. Lee, H. Kim, I. Degani, et al., “Empowering the On-Site Detection of Nucleic Acids by Integrating CRISPR and Digital Signal Processing,” Nature Communications 15 (2024): 6271, https://doi.org/10.1038/s41467-024-50588-3.

[200] J. Zhao, D. Kong, G. Zhang, et al., “An Efficient CRISPR/Cas Cooperative Shearing Platform for Clinical Diagnostics Applications,” Angewandte Chemie International Edition in English 63 (2024): e202411705, https://doi.org/10.1002/anie.202411705.

[201] L. Yang, B. Guo, Y. Wang, et al., “Pyrococcus furiosus Argonaute Combined with Recombinase Polymerase Amplification for Rapid and Sensitive Detection of Enterocytozoon hepatopenaei,” Journal of Agricultural and Food Chemistry 71, no. 1 (2023): 944–951.

[202] J. Qiao, J. Jia, W. Peng, et al., “Rapid, On-Site and Yes-or-No Detection of Salmonella Typhimurium in Foods Using Argonaute-Enabled Assay,” Sensors and Actuators B: Chemical 404 (2024): 135263, https://doi.org/10.1016/j.snb.2023.135263.

[203] J. Wen, M. Han, N. Feng, et al., “A Digital Platform for One-Pot Signal Enhanced Foodborne Pathogen Detection Based on Mesophilic Argonaute-Driven Polydisperse Microdroplet Reactors and Machine Learning,” Chemical Engineering Journal 482 (2024): 148845, https://doi.org/10.1016/j.cej.2024.148845.

[204] G. Xun, Q. Liu, Y. Chong, et al., “Argonaute With Stepwise Endonuclease Activity Promotes Specific and Multiplex Nucleic Acid Detection,” Bioresources and Bioprocessing 8 (2021): 46, https://doi.org/10.1186/s40643-021-00401-6.

[205] H. Altae-Tran, S. Kannan, F. E. Demircioglu, et al., “The Widespread IS200/IS605 Transposon Family Encodes Diverse Programmable RNA-Guided Endonucleases,” Science 374 (2021): 57–65, https://doi.org/10.1126/science.abj6856.

[206] T. Karvelis, G. Druteika, G. Bigelyte, et al., “Transposon-Associated TnpB is a Programmable RNA-Guided DNA Endonuclease,” Nature 599 (2021): 692–696, https://doi.org/10.1038/s41586-021-04058-1.

[207] K. Kato, S. Okazaki, S. Kannan, et al., “Structure of the IscB–ωRNA Ribonucleoprotein Complex, the Likely Ancestor of CRISPR-Cas9,” Nature Communications 13 (2022): 6719, https://doi.org/10.1038/s41467-022-34378-3.

[208] N. Jia and D. J. Patel, “Structure-Based Evolutionary Relationship Between IscB and Cas9,” Cell Research 32 (2022): 875–877, https://doi.org/10.1038/s41422-022-00709-8.

[209] S. Hirano, K. Kappel, H. Altae-Tran, et al., “Structure of the OMEGA Nickase IsrB in Complex With ωRNA and Target DNA,” Nature 610 (2022): 575–581, https://doi.org/10.1038/s41586-022-05324-6.

[210] M. Saito, P. Xu, G. Faure, et al., “Fanzor is a Eukaryotic Programmable RNA-Guided Endonuclease,” Nature 620 (2023): 660–668, https://doi.org/10.1038/s41586-023-06356-2.

[211] P. Xu, M. Saito, G. Faure, et al., “Structural Insights Into the Diversity and DNA Cleavage Mechanism of Fanzor,” Cell 187 (2024): 5238–5252.e20, https://doi.org/10.1016/j.cell.2024.07.050.

[212] M. G. Durrant, N. T. Perry, J. J. Pai, et al., “Bridge RNAs Direct Programmable Recombination of Target and Donor DNA,” Nature 630 (2024): 984–993, https://doi.org/10.1038/s41586-024-07552-4.

[213] M. Hiraizumi, N. T. Perry, M. G. Durrant, et al., “Structural Mechanism of Bridge RNA-Guided Recombination,” Nature 630 (2024): 994–1002, https://doi.org/10.1038/s41586-024-07570-2.

[214] Q. Xiao, G. Li, D. Han, et al., “Engineered IscB–ωRNA System With Expanded Target Range for Base Editing,” Nature Chemical Biology 21 (2024): 100–108, https://doi.org/10.1038/s41589-024-01706-1.

[215] L. Han, Y. Hu, Q. Mo, et al., “Engineering Miniature IscB Nickase for Robust Base Editing With Broad Targeting Range,” Nature Chemical Biology 20 (2024): 1629–1639, https://doi.org/10.1038/s41589-024-01670-w.

[216] H. Yan, X. Tan, S. Zou, et al., “Assessing and Engineering the IscB–ωRNA System for Programmed Genome Editing,” Nature Chemical Biology 20 (2024): 1617–1628, https://doi.org/10.1038/s41589-024-01669-3.

[217] D. Han, Q. Xiao, Y. Wang, et al., “Development of Miniature Base Editors Using Engineered IscB Nickase,” Nature Methods 20 (2023): 1029–1036, https://doi.org/10.1038/s41592-023-01898-9.

[218] K. F. Marquart, N. Mathis, A. Mollaysa, et al., “Effective Genome Editing With an Enhanced ISDra2 TnpB System and Deep Learning-Predicted ωRNAs,” Nature Methods 21 (2024): 2084–2093, https://doi.org/10.1038/s41592-024-02418-z.

[219] Z. Li, R. Guo, X. Sun, et al., “Engineering a Transposon-Associated TnpB-ωRNA System for Efficient Gene Editing and Phenotypic Correction of a Tyrosinaemia Mouse Model,” Nature Communications 15 (2024): 831, https://doi.org/10.1038/s41467-024-45197-z.

[220] X. Feng, R. Xu, J. Liao, et al., “Flexible TAM Requirement of TnpB Enables Efficient Single-Nucleotide Editing With Expanded Targeting Scope,” Nature Communications 15 (2024): 3464, https://doi.org/10.1038/s41467-024-47697-4.

[221] Y. Xu, T. Liu, J. Wang, et al., “Reprogramming an RNA-Guided Archaeal TnpB Endonuclease for Genome Editing,” Cell Discovery 9 (2023): 112, https://doi.org/10.1038/s41421-023-00615-2.

[222] G. Otto, “A Vast Potential Genome Editor Toolbox,” Nature Reviews Genetics 22 (2021): 747, https://doi.org/10.1038/s41576-021-00429-6.

[223] J. Listgarten, M. Weinstein, B. P. Kleinstiver, et al., “Prediction of Off-Target Activities for the End-to-End Design of CRISPR Guide RNAs,” Nature Biomedical Engineering 2 (2018): 38–47, https://doi.org/10.1038/s41551-017-0178-6.

[224] H.-H. Wessels, A. Stirn, A. Méndez-Mancilla, et al., “Prediction of On-Target and Off-Target Activity of CRISPR–Cas13d Guide RNAs Using Deep Learning,” Nature Biotechnology 42 (2024): 628–637, https://doi.org/10.1038/s41587-023-01830-8.

[225] T. Biancalani, G. Scalia, L. Buffoni, et al., “Deep Learning and Alignment of Spatially Resolved Single-Cell Transcriptomes With Tangram,” Nature Methods 18 (2021): 1352–1362, https://doi.org/10.1038/s41592-021-01264-7.

[226] H. Hong, S. Jiang, H. Li, et al., “DeepHiC: A Generative Adversarial Network for Enhancing Hi-C Data Resolution,” PLoS Computational Biology 16 (2020): e1007287, https://doi.org/10.1371/journal.pcbi.1007287.

RIGHTS & PERMISSIONS

2026 The Author(s). Exploration published by Henan University and John Wiley & Sons Australia, Ltd.