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dc.contributor.authorLiao, Chunyu
dc.contributor.authorSlotkowski, Rebecca A
dc.contributor.authorAchmedov, Tatjana
dc.contributor.authorBeisel, Chase L
dc.date.accessioned2018-12-04T13:53:22Z
dc.date.available2018-12-04T13:53:22Z
dc.date.issued2018-10-12
dc.identifier.issn1555-8584
dc.identifier.pmid30252595
dc.identifier.doi10.1080/15476286.2018.1526537
dc.identifier.urihttp://hdl.handle.net/10033/621601
dc.description.abstractThe Class 2 Type V-A CRISPR effector protein Cas12a/Cpf1 has gained widespread attention in part because of the ease in achieving multiplexed genome editing, gene regulation, and DNA detection. Multiplexing derives from the ability of Cas12a alone to generate multiple guide RNAs from a transcribed CRISPR array encoding alternating conserved repeats and targeting spacers. While array design has focused on how to optimize guide-RNA sequences, little attention has been paid to sequences outside of the CRISPR array. Here, we show that a structured hairpin located immediately downstream of the 3' repeat interferes with utilization of the adjacent encoded guide RNA by Francisella novicida (Fn)Cas12a. We first observed that a synthetic Rho-independent terminator immediately downstream of an array impaired DNA cleavage based on plasmid clearance in E. coli and DNA cleavage in a cell-free transcription-translation (TXTL) system. TXTL-based cleavage assays further revealed that inhibition was associated with incomplete processing of the transcribed CRISPR array and could be attributed to the stable hairpin formed by the terminator. We also found that the inhibitory effect partially extended to upstream spacers in a multi-spacer array. Finally, we found that removing the terminal repeat from the array increased the inhibitory effect, while replacing this repeat with an unprocessable terminal repeat from a native FnCas12a array restored cleavage activity directed by the adjacent encoded guide RNA. Our study thus revealed that sequences surrounding a CRISPR array can interfere with the function of a CRISPR nuclease, with implications for the design and evolution of CRISPR arrays.en_US
dc.rightsAttribution-NonCommercial-ShareAlike 4.0 International*
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.subjectCRISPRen_US
dc.subjectCpf1en_US
dc.subjectRNA structureen_US
dc.subjectTXTLen_US
dc.subjectterminatoren_US
dc.titleThe Francisella novicida Cas12a is sensitive to the structure downstream of the terminal repeat in CRISPR arrays.en_US
dc.typeArticleen_US
dc.contributor.departmentHIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.en_US
dc.source.journaltitleRNA biology


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Except where otherwise noted, this item's license is described as Attribution-NonCommercial-ShareAlike 4.0 International