CRISPR-Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes

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dc.contributor.authorTong, Yaojunko
dc.contributor.authorWhitford, Christopher M.ko
dc.contributor.authorBlin, Kaiko
dc.contributor.authorJorgensen, Tue S.ko
dc.contributor.authorWeber, Tilmannko
dc.contributor.authorLee, Sang Yupko
dc.date.accessioned2020-08-13T07:55:19Z-
dc.date.available2020-08-13T07:55:19Z-
dc.date.created2020-07-27-
dc.date.created2020-07-27-
dc.date.issued2020-08-
dc.identifier.citationNATURE PROTOCOLS, v.15, no.8, pp.2470 - 2502-
dc.identifier.issn1754-2189-
dc.identifier.urihttp://hdl.handle.net/10203/275823-
dc.description.abstractStreptomycetes are prominent sources of bioactive natural products, but metabolic engineering of the natural products of these organisms is greatly hindered by relatively inefficient genetic manipulation approaches. New advances in genome editing techniques, particularly CRISPR-based tools, have revolutionized genetic manipulation of many organisms, including actinomycetes. We have developed a comprehensive CRISPR toolkit that includes several variations of 'classic' CRISPR-Cas9 systems, along with CRISPRi and CRISPR-base editing systems (CRISPR-BEST) for streptomycetes. Here, we provide step-by-step protocols for designing and constructing the CRISPR plasmids, transferring these plasmids to the target streptomycetes, and identifying correctly edited clones. Our CRISPR toolkit can be used to generate random-sized deletion libraries, introduce small indels, generate in-frame deletions of specific target genes, reversibly suppress gene transcription, and substitute single base pairs in streptomycete genomes. Furthermore, the toolkit includes a Csy4-based multiplexing option to introduce multiple edits in a single experiment. The toolkit can be easily extended to other actinomycetes. With our protocol, it takes <10 d to inactivate a target gene, which is much faster than alternative protocols. This protocol offers a CRISPR-based toolkit, including several variants of 'classic' CRISPR-Cas9, along with CRISPRi and CRISPR-base editing systems (CRISPR-BEST) for genome editing in streptomycetes.-
dc.languageEnglish-
dc.publisherNATURE PUBLISHING GROUP-
dc.titleCRISPR-Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes-
dc.typeArticle-
dc.identifier.wosid000547241700001-
dc.identifier.scopusid2-s2.0-85087673586-
dc.type.rimsART-
dc.citation.volume15-
dc.citation.issue8-
dc.citation.beginningpage2470-
dc.citation.endingpage2502-
dc.citation.publicationnameNATURE PROTOCOLS-
dc.identifier.doi10.1038/s41596-020-0339-z-
dc.contributor.localauthorLee, Sang Yup-
dc.contributor.nonIdAuthorTong, Yaojun-
dc.contributor.nonIdAuthorWhitford, Christopher M.-
dc.contributor.nonIdAuthorBlin, Kai-
dc.contributor.nonIdAuthorJorgensen, Tue S.-
dc.contributor.nonIdAuthorWeber, Tilmann-
dc.description.isOpenAccessN-
dc.type.journalArticleArticle-
dc.subject.keywordPlusNATURAL-PRODUCTS-
dc.subject.keywordPlusGENOMIC DNA-
dc.subject.keywordPlusSEQUENCE-
dc.subject.keywordPlusBASE-
dc.subject.keywordPlusBIOSYNTHESIS-
dc.subject.keywordPlusMUTATIONS-
dc.subject.keywordPlusCLUSTER-
dc.subject.keywordPlusDESIGN-
dc.subject.keywordPlusSGRNAS-
dc.subject.keywordPlusWEB-
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CBE-Journal Papers(저널논문)
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