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dc.contributor.authorMaxwell, Colin S
dc.contributor.authorJacobsen, Thomas
dc.contributor.authorMarshall, Ryan
dc.contributor.authorNoireaux, Vincent
dc.contributor.authorBeisel, Chase L
dc.date.accessioned2020-07-09T11:02:40Z
dc.date.available2020-07-09T11:02:40Z
dc.date.issued2018-02-24
dc.identifier.citationMethods. 2018;143:48-57. doi:10.1016/j.ymeth.2018.02.016.en_US
dc.identifier.pmid29486239
dc.identifier.doi10.1016/j.ymeth.2018.02.016
dc.identifier.urihttp://hdl.handle.net/10033/622345
dc.description.abstractThe RNA-guided nucleases derived from the CRISPR-Cas systems in bacteria and archaea have found numerous applications in biotechnology, including genome editing, imaging, and gene regulation. However, the discovery of novel Cas nucleases has outpaced their characterization and subsequent exploitation. A key step in characterizing Cas nucleases is determining which protospacer-adjacent motif (PAM) sequences they recognize. Here, we report advances to an in vitro method based on an E. coli cell-free transcription-translation system (TXTL) to rapidly elucidate PAMs recognized by Cas nucleases. The method obviates the need for cloning Cas nucleases or gRNAs, does not require the purification of protein or RNA, and can be performed in less than a day. To advance our previously published method, we incorporated an internal GFP cleavage control to assess the extent of library cleavage as well as Sanger sequencing of the cleaved library to assess PAM depletion prior to next-generation sequencing. We also detail the methods needed to construct all relevant DNA constructs, and how to troubleshoot the assay. We finally demonstrate the technique by determining PAM sequences recognized by the Neisseria meningitidis Cas9, revealing subtle sequence requirements of this highly specific PAM. The overall method offers a rapid means to identify PAMs recognized by diverse CRISPR nucleases, with the potential to greatly accelerate our ability to characterize and harness novel CRISPR nucleases across their many uses.en_US
dc.language.isoenen_US
dc.publisherElsevieren_US
dc.rightsAttribution-NonCommercial-ShareAlike 4.0 International*
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.subjectCas12aen_US
dc.subjectCas9en_US
dc.subjectPAMen_US
dc.subjectTXTLen_US
dc.subjectcrRNAen_US
dc.subjectsgRNAen_US
dc.titleA detailed cell-free transcription-translation-based assay to decipher CRISPR protospacer-adjacent motifs.en_US
dc.typeArticleen_US
dc.typeOtheren_US
dc.identifier.eissn1095-9130
dc.contributor.departmentHIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.en_US
dc.identifier.journalMethods (San Diego, Calif.)en_US
dc.source.volume143
dc.source.beginpage48
dc.source.endpage57
refterms.dateFOA2020-07-09T11:02:41Z
dc.source.journaltitleMethods (San Diego, Calif.)
dc.source.countryUnited States
dc.source.countryUnited States
dc.source.countryUnited States


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