Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST

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dc.contributor.authorTong, Yaojunko
dc.contributor.authorWhitford, Christopher M.ko
dc.contributor.authorRobertsen, Helene L.ko
dc.contributor.authorBlin, Kaiko
dc.contributor.authorJorgensen, Tue S.ko
dc.contributor.authorKlitgaard, Andreas K.ko
dc.contributor.authorGren, Tetianako
dc.contributor.authorJiang, Xinglinko
dc.contributor.authorWeber, Tilmannko
dc.contributor.authorLee, Sang Yupko
dc.date.accessioned2019-10-31T06:20:41Z-
dc.date.available2019-10-31T06:20:41Z-
dc.date.created2019-10-31-
dc.date.created2019-10-31-
dc.date.created2019-10-31-
dc.date.issued2019-10-
dc.identifier.citationPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, v.116, no.41, pp.20366 - 20375-
dc.identifier.issn0027-8424-
dc.identifier.urihttp://hdl.handle.net/10203/268088-
dc.description.abstractStreptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide-resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different Streptomyces species. Particularly in nonmodel actinomycete Streptomyces collinus Tu365, CRISPR-cBEST efficiently inactivated the 2 copies of kirN gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the proto-spacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.-
dc.languageEnglish-
dc.publisherNATL ACAD SCIENCES-
dc.titleHighly efficient DSB-free base editing for streptomycetes with CRISPR-BEST-
dc.typeArticle-
dc.identifier.wosid000489770700025-
dc.identifier.scopusid2-s2.0-85073071217-
dc.type.rimsART-
dc.citation.volume116-
dc.citation.issue41-
dc.citation.beginningpage20366-
dc.citation.endingpage20375-
dc.citation.publicationnamePROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-
dc.identifier.doi10.1073/pnas.1913493116-
dc.contributor.localauthorLee, Sang Yup-
dc.contributor.nonIdAuthorTong, Yaojun-
dc.contributor.nonIdAuthorWhitford, Christopher M.-
dc.contributor.nonIdAuthorRobertsen, Helene L.-
dc.contributor.nonIdAuthorBlin, Kai-
dc.contributor.nonIdAuthorJorgensen, Tue S.-
dc.contributor.nonIdAuthorKlitgaard, Andreas K.-
dc.contributor.nonIdAuthorGren, Tetiana-
dc.contributor.nonIdAuthorJiang, Xinglin-
dc.contributor.nonIdAuthorWeber, Tilmann-
dc.description.isOpenAccessY-
dc.type.journalArticleArticle-
dc.subject.keywordAuthorstreptomycetes-
dc.subject.keywordAuthorCRISPR base editor-
dc.subject.keywordAuthorcytidine deaminase-
dc.subject.keywordAuthoradenosine deaminase-
dc.subject.keywordAuthorgenome editing-
dc.subject.keywordPlusCOMPLETE GENOME SEQUENCE-
dc.subject.keywordPlusDNA-REPAIR-
dc.subject.keywordPlusRESISTANCE GENE-
dc.subject.keywordPlusCLONING-
dc.subject.keywordPlusEXPRESSION-
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