{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T19:19:46Z","timestamp":1789759186201,"version":"4.0.1"},"reference-count":37,"publisher":"Springer Science and Business Media LLC","issue":"10","license":[{"start":{"date-parts":[[2025,2,24]],"date-time":"2025-02-24T00:00:00Z","timestamp":1740355200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/www.springernature.com\/gp\/researchers\/text-and-data-mining"},{"start":{"date-parts":[[2025,2,24]],"date-time":"2025-02-24T00:00:00Z","timestamp":1740355200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.springernature.com\/gp\/researchers\/text-and-data-mining"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Nat Protoc"],"published-print":{"date-parts":[[2025,10]]},"DOI":"10.1038\/s41596-024-01138-0","type":"journal-article","created":{"date-parts":[[2025,2,24]],"date-time":"2025-02-24T04:02:29Z","timestamp":1740369749000},"page":"2942-2959","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Circular Vectors as an efficient, fully synthetic, cell-free approach for preparing small circular DNA as a plasmid substitute for guide RNA expression in CRISPR\u2013Cas9 genome editing"],"prefix":"10.1038","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1893-199X","authenticated-orcid":false,"given":"Roman Teo","family":"Oliynyk","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6232-9969","authenticated-orcid":false,"given":"George M.","family":"Church","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,2,24]]},"reference":[{"key":"1138_CR1","doi-asserted-by":"crossref","unstructured":"Byrne, S. M., Mali, P. & Church, G. M. in Methods in Enzymology Vol. 546 (eds Doudna, J. A. & Sontheimer, E. J.) 119\u2013138 (Academic, 2014).","DOI":"10.1016\/B978-0-12-801185-0.00006-4"},{"key":"1138_CR2","unstructured":"Doghaither, H. A. & Gull, M. in Plasmid (ed. Gull, M.) Ch. 5 (IntechOpen, 2019)."},{"key":"1138_CR3","doi-asserted-by":"publisher","first-page":"5532","DOI":"10.7150\/thno.43465","volume":"10","author":"AK Fajrial","year":"2020","unstructured":"Fajrial, A. K., He, Q. Q., Wirusanti, N. I., Slansky, J. E. & Ding, X. A review of emerging physical transfection methods for CRISPR\/Cas9-mediated gene editing. Theranostics 10, 5532\u20135549 (2020).","journal-title":"Theranostics"},{"key":"1138_CR4","doi-asserted-by":"publisher","first-page":"a023754","DOI":"10.1101\/cshperspect.a023754","volume":"8","author":"T Gaj","year":"2016","unstructured":"Gaj, T., Sirk, S. J., Shui, S.-L. & Liu, J. Genome-editing technologies: principles and applications. Cold Spring Harb. Perspect. Biol. 8, a023754 (2016).","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"1138_CR5","doi-asserted-by":"publisher","first-page":"171","DOI":"10.1038\/nprot.2011.431","volume":"7","author":"NE Sanjana","year":"2012","unstructured":"Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nat. Protoc. 7, 171\u2013192 (2012).","journal-title":"Nat. Protoc."},{"key":"1138_CR6","doi-asserted-by":"publisher","first-page":"2281","DOI":"10.1038\/nprot.2013.143","volume":"8","author":"FA Ran","year":"2013","unstructured":"Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281\u20132308 (2013).","journal-title":"Nat. Protoc."},{"key":"1138_CR7","doi-asserted-by":"publisher","first-page":"819","DOI":"10.1126\/science.1231143","volume":"339","author":"L Cong","year":"2013","unstructured":"Cong, L. et al. Multiplex genome engineering using CRISPR\/Cas systems. Science 339, 819\u2013823 (2013).","journal-title":"Science"},{"key":"1138_CR8","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-12281-8","volume":"10","author":"D Wang","year":"2019","unstructured":"Wang, D. et al. Optimized CRISPR guide RNA design for two high-fidelity Cas9 variants by deep learning. Nat. Commun. 10, 4284 (2019).","journal-title":"Nat. Commun."},{"key":"1138_CR9","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1016\/j.jconrel.2017.09.012","volume":"266","author":"C Liu","year":"2017","unstructured":"Liu, C., Zhang, L., Liu, H. & Cheng, K. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. J. Control. Release 266, 17\u201326 (2017).","journal-title":"J. Control. Release"},{"key":"1138_CR10","doi-asserted-by":"publisher","first-page":"149","DOI":"10.1038\/s41586-019-1711-4","volume":"576","author":"AV Anzalone","year":"2019","unstructured":"Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149\u2013157 (2019).","journal-title":"Nature"},{"key":"1138_CR11","doi-asserted-by":"publisher","first-page":"5635","DOI":"10.1016\/j.cell.2021.09.018","volume":"184","author":"PJ Chen","year":"2021","unstructured":"Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635\u20135652.e29 (2021).","journal-title":"Cell"},{"key":"1138_CR12","doi-asserted-by":"publisher","first-page":"268","DOI":"10.1038\/s41556-021-00836-1","volume":"24","author":"C Wang","year":"2022","unstructured":"Wang, C. et al. dCas9-based gene editing for cleavage-free genomic knock-in of long sequences. Nat. Cell Biol. 24, 268\u2013278 (2022).","journal-title":"Nat. Cell Biol."},{"key":"1138_CR13","doi-asserted-by":"publisher","first-page":"36","DOI":"10.1016\/j.omtn.2017.11.006","volume":"10","author":"Z Gao","year":"2018","unstructured":"Gao, Z., Herrera-Carrillo, E. & Berkhout, B. Delineation of the exact transcription termination signal for Type 3 Polymerase III. Mol. Ther. Nucleic Acids 10, 36\u201344 (2018).","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"1138_CR14","doi-asserted-by":"publisher","first-page":"311","DOI":"10.1007\/s12033-022-00567-0","volume":"65","author":"VE Hillary","year":"2023","unstructured":"Hillary, V. E. & Ceasar, S. A. A review on the mechanism and applications of CRISPR\/Cas9\/Cas12\/Cas13\/Cas14 proteins utilized for genome engineering. Mol. Biotechnol. 65, 311\u2013325 (2023).","journal-title":"Mol. Biotechnol."},{"key":"1138_CR15","doi-asserted-by":"publisher","first-page":"233","DOI":"10.1016\/j.stem.2015.06.001","volume":"17","author":"Y Chen","year":"2015","unstructured":"Chen, Y. et al. Engineering human stem cell lines with inducible gene knockout using CRISPR\/Cas9. Cell Stem Cell 17, 233\u2013244 (2015).","journal-title":"Cell Stem Cell"},{"key":"1138_CR16","doi-asserted-by":"publisher","first-page":"e3647","DOI":"10.1371\/journal.pone.0003647","volume":"3","author":"C Engler","year":"2008","unstructured":"Engler, C., Kandzia, R. & Marillonnet, S. A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3, e3647 (2008).","journal-title":"PLoS ONE"},{"key":"1138_CR17","doi-asserted-by":"publisher","first-page":"10785","DOI":"10.1093\/nar\/gkab792","volume":"49","author":"F Adikusuma","year":"2021","unstructured":"Adikusuma, F. et al. Optimized nickase- and nuclease-based prime editing in human and mouse cells. Nucleic Acids Res. 49, 10785\u201310795 (2021).","journal-title":"Nucleic Acids Res."},{"key":"1138_CR18","doi-asserted-by":"publisher","first-page":"343","DOI":"10.1038\/nmeth.1318","volume":"6","author":"DG Gibson","year":"2009","unstructured":"Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343\u2013345 (2009).","journal-title":"Nat. Methods"},{"key":"1138_CR19","doi-asserted-by":"publisher","unstructured":"Bai, X. et al. Prevalence of errors in lab-made plasmids across the globe. Preprint at bioRxiv https:\/\/doi.org\/10.1101\/2024.06.17.596931 (2024).","DOI":"10.1101\/2024.06.17.596931"},{"key":"1138_CR20","doi-asserted-by":"publisher","first-page":"1393","DOI":"10.1038\/s42003-022-04363-z","volume":"5","author":"RT Oliynyk","year":"2022","unstructured":"Oliynyk, R. T. & Church, G. M. Efficient modification and preparation of circular DNA for expression in cell culture. Commun. Biol. 5, 1393 (2022).","journal-title":"Commun. Biol."},{"key":"1138_CR21","doi-asserted-by":"publisher","unstructured":"Oliynyk, R. T., Mahas, A., Karpinski, E. & Church, G. M. Plasmid2MC: efficient cell-free recombination of plasmids into high-purity minicircle DNA for use in genome editing applications. Preprint at bioRxiv https:\/\/doi.org\/10.1101\/2024.07.08.602524 (2024).","DOI":"10.1101\/2024.07.08.602524"},{"key":"1138_CR22","doi-asserted-by":"publisher","first-page":"1341","DOI":"10.1038\/sj.gt.3300540","volume":"4","author":"A Darquet","year":"1997","unstructured":"Darquet, A., Cameron, B., Wils, P., Scherman, D. & Crouzet, J. A new DNA vehicle for nonviral gene delivery: supercoiled minicircle. Gene Ther. 4, 1341\u20131349 (1997).","journal-title":"Gene Ther."},{"key":"1138_CR23","doi-asserted-by":"publisher","first-page":"209","DOI":"10.1038\/sj.gt.3300816","volume":"6","author":"A-M Darquet","year":"1999","unstructured":"Darquet, A.-M. et al. Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer. Gene Ther. 6, 209\u2013218 (1999).","journal-title":"Gene Ther."},{"key":"1138_CR24","doi-asserted-by":"publisher","first-page":"82","DOI":"10.1089\/nat.2020.0904","volume":"31","author":"D Eus\u00e9bio","year":"2021","unstructured":"Eus\u00e9bio, D. et al. The performance of minicircle DNA versus parental plasmid in p53 gene delivery into HPV-18-infected cervical cancer cells. Nucleic Acid Ther. 31, 82\u201391 (2021).","journal-title":"Nucleic Acid Ther."},{"key":"1138_CR25","doi-asserted-by":"publisher","first-page":"144","DOI":"10.1038\/nature20565","volume":"540","author":"K Suzuki","year":"2016","unstructured":"Suzuki, K. et al. In vivo genome editing via CRISPR\/Cas9 mediated homology-independent targeted integration. Nature 540, 144\u2013149 (2016).","journal-title":"Nature"},{"key":"1138_CR26","doi-asserted-by":"publisher","first-page":"157","DOI":"10.1038\/s10038-017-0352-4","volume":"63","author":"K Suzuki","year":"2018","unstructured":"Suzuki, K. & Izpisua Belmonte, J. C. In vivo genome editing via the HITI method as a tool for gene therapy. J. Hum. Genet. 63, 157\u2013164 (2018).","journal-title":"J. Hum. Genet."},{"key":"1138_CR27","doi-asserted-by":"publisher","first-page":"541","DOI":"10.1038\/s41592-023-01809-y","volume":"20","author":"TWJ Gadella","year":"2023","unstructured":"Gadella, T. W. J. et al. mScarlet3: a brilliant and fast-maturing red fluorescent protein. Nat. Methods 20, 541\u2013545 (2023).","journal-title":"Nat. Methods"},{"key":"1138_CR28","doi-asserted-by":"publisher","first-page":"883","DOI":"10.1038\/s41587-020-0453-z","volume":"38","author":"MF Richter","year":"2020","unstructured":"Richter, M. F. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat. Biotechnol. 38, 883\u2013891 (2020).","journal-title":"Nat. Biotechnol."},{"key":"1138_CR29","doi-asserted-by":"publisher","first-page":"023189","DOI":"10.1103\/PhysRevResearch.6.023189","volume":"6","author":"M Panoukidou","year":"2024","unstructured":"Panoukidou, M. et al. Runaway transition in irreversible polymer condensation with cyclization. Phys. Rev. Res. 6, 023189 (2024).","journal-title":"Phys. Rev. Res."},{"key":"1138_CR30","doi-asserted-by":"publisher","first-page":"171","DOI":"10.1016\/0022-2836(75)90189-8","volume":"96","author":"A Dugaiczyk","year":"1975","unstructured":"Dugaiczyk, A., Boyer, H. W. & Goodman, H. M. Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures. J. Mol. Biol. 96, 171\u2013184 (1975).","journal-title":"J. Mol. Biol."},{"key":"1138_CR31","doi-asserted-by":"publisher","first-page":"542","DOI":"10.1186\/s12859-018-2585-4","volume":"19","author":"G-H Hwang","year":"2018","unstructured":"Hwang, G.-H. et al. Web-based design and analysis tools for CRISPR base editing. BMC Bioinform. 19, 542 (2018).","journal-title":"BMC Bioinform."},{"key":"1138_CR32","doi-asserted-by":"publisher","first-page":"W499","DOI":"10.1093\/nar\/gkab319","volume":"49","author":"G-H Hwang","year":"2021","unstructured":"Hwang, G.-H. et al. PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing. Nucleic Acids Res. 49, W499\u2013W504 (2021).","journal-title":"Nucleic Acids Res."},{"key":"1138_CR33","doi-asserted-by":"publisher","first-page":"190","DOI":"10.1038\/s41551-020-00622-8","volume":"5","author":"RD Chow","year":"2021","unstructured":"Chow, R. D., Chen, J. S., Shen, J. & Chen, S. A web tool for the design of prime-editing guide RNAs. Nat. Biomed. Eng. 5, 190\u2013194 (2021).","journal-title":"Nat. Biomed. Eng."},{"key":"1138_CR34","doi-asserted-by":"publisher","first-page":"1261","DOI":"10.1038\/s42256-023-00739-w","volume":"5","author":"F Liu","year":"2023","unstructured":"Liu, F. et al. Design of prime-editing guide RNAs with deep transfer learning. Nat. Mach. Intell. 5, 1261\u20131274 (2023).","journal-title":"Nat. Mach. Intell."},{"key":"1138_CR35","doi-asserted-by":"publisher","first-page":"402","DOI":"10.1038\/s41587-021-01039-7","volume":"40","author":"JW Nelson","year":"2022","unstructured":"Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 40, 402\u2013410 (2022).","journal-title":"Nat. Biotechnol."},{"key":"1138_CR36","first-page":"24","volume":"44","author":"Y-T Kim","year":"2012","unstructured":"Kim, Y.-T. et al. Effects of storage buffer and temperature on the integrity of human DNA. Korean J. Clin. Lab. Sci. 44, 24\u201330 (2012).","journal-title":"Korean J. Clin. Lab. Sci."},{"key":"1138_CR37","doi-asserted-by":"publisher","first-page":"1312","DOI":"10.1038\/nprot.2008.112","volume":"3","author":"UH Frey","year":"2008","unstructured":"Frey, U. H., Bachmann, H. S., Peters, J. & Siffert, W. PCR-amplification of GC-rich regions: \u2018slowdown PCR\u2019. Nat. Protoc. 3, 1312\u20131317 (2008).","journal-title":"Nat. Protoc."}],"container-title":["Nature Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41596-024-01138-0.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41596-024-01138-0","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41596-024-01138-0.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T05:43:00Z","timestamp":1785476580000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41596-024-01138-0"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2,24]]},"references-count":37,"journal-issue":{"issue":"10","published-print":{"date-parts":[[2025,10]]}},"alternative-id":["1138"],"URL":"https:\/\/doi.org\/10.1038\/s41596-024-01138-0","relation":{},"ISSN":["1754-2189","1750-2799"],"issn-type":[{"value":"1754-2189","type":"print"},{"value":"1750-2799","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,2,24]]},"assertion":[{"value":"4 August 2024","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"19 December 2024","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"24 February 2025","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"G.M.C. disclosures are available at\n                      \n                      . R.T.O. declares no competing interests.","order":1,"name":"Ethics","label":"Competing interests","group":{"name":"EthicsHeading","label":"Ethics"}}]}}