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dc.contributor.authorWandera, Katharina G
dc.contributor.authorCollins, Scott P
dc.contributor.authorWimmer, Franziska
dc.contributor.authorMarshall, Ryan
dc.contributor.authorNoireaux, Vincent
dc.contributor.authorBeisel, Chase L
dc.date.accessioned2019-07-10T14:37:17Z
dc.date.available2019-07-10T14:37:17Z
dc.date.issued2019-05-21
dc.identifier.citationMethods. 2019 May 21. pii: S1046-2023(19)30001-5. doi: 10.1016/j.ymeth.2019.05.014.en_US
dc.identifier.issn1095-9130
dc.identifier.pmid31121300
dc.identifier.doi10.1016/j.ymeth.2019.05.014
dc.identifier.urihttp://hdl.handle.net/10033/621858
dc.description.abstractThe characterization of CRISPR-Cas immune systems in bacteria was quickly followed by the discovery of anti-CRISPR proteins (Acrs) in bacteriophages. These proteins block different steps of CRISPR-based immunity and, as some inhibit Cas nucleases, can offer tight control over CRISPR technologies. While Acrs have been identified against a few CRISPR-Cas systems, likely many more await discovery and application. Here, we report a rapid and scalable method for characterizing putative Acrs against Cas nucleases using an E. coli-derived cell-free transcription-translation system. Using known Acrs against type II Cas9 nucleases as models, we demonstrate how the method can be used to measure the inhibitory activity of individual Acrs in under two days. We also show how the method can overcome non-specific inhibition of gene expression observed for some Acrs. In total, the method should accelerate the interrogation and application of Acrs as CRISPR-Cas inhibitors.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.subjectAnti-CRISPR proteinsen_US
dc.subjectCas9en_US
dc.subjectGenome editingen_US
dc.subjectTXTLen_US
dc.subjectsgRNAen_US
dc.titleAn enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system.en_US
dc.typeArticleen_US
dc.contributor.departmentHIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.en_US
dc.identifier.journalMethodsen_US
dc.source.journaltitleMethods (San Diego, Calif.)


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