ProtocolPublished: 24 February 2025Roman Teo Oliynyk ORCID: orcid.org/0000-0003-1893-199X1,2 &George M. Church ORCID: orcid.org/0000-0001-6232-99691,3 Nature Protocols (2025)Cite this articleMetrics detailsSubjectsCRISPR-Cas9 genome editingGenetics researchSingle-molecule biophysicsAbstractRobust expression of guide RNA (gRNA) is essential for successful implementation of CRISPR–Cas9 genome-editing methods. The gRNA components, such as an RNA polymerase promoter followed by the gRNA coding sequence and an RNA polymerase terminator sequence, and the Cas9 protein are expressed either via an all-in-one plasmid or separate dedicated plasmids. The preparation of such plasmids involves a laborious multi-day process of DNA assembly, bacterial cloning, validation, purification and sequencing. Our Circular Vector (CV) protocol introduces an efficient, fully synthetic, cell-free approach for preparing gRNA expression templates suitable for transfection, marking a significant advancement over traditional plasmid-based approaches. This protocol consists of the circularization and purification of linear double-stranded DNA (dsDNA) containing gRNA expression elements into compact, bacterial-backbone-free circular DNA expression vectors in as little as 3 h. We provide a guide to the design of the dsDNA template coding for gRNA elements for CRISPR–Cas9 base and prime editing, along with step-by-step instructions for the efficient preparation of gRNA-expressing CVs. In addition to rapid preparation, CVs created via this protocol offer several key advantages: a compact size, absence of a bacterial backbone, absence of bacterial endotoxins and no contamination by bacterial RNA or DNA fragments. These features make gRNA-expressing CVs a superior choice over plasmid-based gRNA expression templates.Key pointsThis Circular Vector (CV) protocol describes a synthetic, cell-free approach for preparing transfection-ready, small, double-stranded DNA templates for gRNA expression for different genome-editing applications.In contrast to traditional plasmid-based expression systems, the CV protocol reduces the time required to prepare expression vectors to several hours, resulting in a gRNA-expression template free of bacterial endotoxin and nucleic acid contaminants.This is a preview of subscription content, access via your institutionAccess optionsAccess Nature and 54 other Nature Portfolio journalsGet Nature+, our best-value online-access subscription24,99 € / 30 dayscancel any timeLearn moreSubscribe to this journalReceive 12 print issues and online access269,00 € per yearonly 22,42 € per issueLearn moreBuy this articlePurchase on SpringerLinkInstant access to full article PDFBuy nowPrices may be subject to local taxes which are calculated during checkoutFig. 1: Overview of the CV preparation steps.Fig. 2: Design of the template dsDNA fragments for the CV preparation steps for gRNA expression.Fig. 3: Circularization yield and concatemer proportions.Data availabilityThe following files are available in Supplementary Data 1 and contain example templates for different genome-editing applications: BaseEditingDemo.gbk, PrimeEditingDemo.gbk, BaseEditingTwistWillAddAdaptersOrder.gbk, PrimeEditingTwistWillAddAdaptersOrder.gbk, BaseEditing-AlternativeManufacturerPrimers.gbk, PrimeEditing-AlternativeManufacturerPrimers.gbk. 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Protoc. 3, 1312–1317 (2008).Article CAS PubMed Google Scholar Download referencesAuthor informationAuthors and AffiliationsDepartment of Genetics, Harvard Medical School, Boston, MA, USARoman Teo Oliynyk & George M. ChurchDepartment of Computer Science, University of Auckland, Auckland, New ZealandRoman Teo OliynykWyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA, USAGeorge M. ChurchAuthorsRoman Teo OliynykView author publicationsYou can also search for this author in PubMed Google ScholarGeorge M. ChurchView author publicationsYou can also search for this author in PubMed Google ScholarContributionsR.T.O. designed and performed the original research on which this protocol is based. R.T.O. and G.M.C. wrote and reviewed the manuscript.Corresponding authorCorrespondence to Roman Teo Oliynyk.Ethics declarationsCompeting interestsG.M.C. disclosures are available at https://arep.med.harvard.edu/gmc/tech.html. R.T.O. declares no competing interests.Peer reviewPeer review informationNature Protocols thanks Duarte Miguel Prazeres, Yiping Qi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Key referenceOliynyk, R. T. et al. Commun. Biol. 5, 1393 (2022): https://doi.org/10.1038/s42003-022-04363-zSupplementary informationSupplementary DataSupplementary DataRights and permissionsSpringer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.Reprints and permissionsAbout this article