DC Field | Value | Language |
---|---|---|
dc.contributor.author | Tong, Yaojun | ko |
dc.contributor.author | Whitford, Christopher M. | ko |
dc.contributor.author | Robertsen, Helene L. | ko |
dc.contributor.author | Blin, Kai | ko |
dc.contributor.author | Jorgensen, Tue S. | ko |
dc.contributor.author | Klitgaard, Andreas K. | ko |
dc.contributor.author | Gren, Tetiana | ko |
dc.contributor.author | Jiang, Xinglin | ko |
dc.contributor.author | Weber, Tilmann | ko |
dc.contributor.author | Lee, Sang Yup | ko |
dc.date.accessioned | 2019-10-31T06:20:41Z | - |
dc.date.available | 2019-10-31T06:20:41Z | - |
dc.date.created | 2019-10-31 | - |
dc.date.created | 2019-10-31 | - |
dc.date.created | 2019-10-31 | - |
dc.date.issued | 2019-10 | - |
dc.identifier.citation | PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, v.116, no.41, pp.20366 - 20375 | - |
dc.identifier.issn | 0027-8424 | - |
dc.identifier.uri | http://hdl.handle.net/10203/268088 | - |
dc.description.abstract | Streptomycetes 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.language | English | - |
dc.publisher | NATL ACAD SCIENCES | - |
dc.title | Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST | - |
dc.type | Article | - |
dc.identifier.wosid | 000489770700025 | - |
dc.identifier.scopusid | 2-s2.0-85073071217 | - |
dc.type.rims | ART | - |
dc.citation.volume | 116 | - |
dc.citation.issue | 41 | - |
dc.citation.beginningpage | 20366 | - |
dc.citation.endingpage | 20375 | - |
dc.citation.publicationname | PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | - |
dc.identifier.doi | 10.1073/pnas.1913493116 | - |
dc.contributor.localauthor | Lee, Sang Yup | - |
dc.contributor.nonIdAuthor | Tong, Yaojun | - |
dc.contributor.nonIdAuthor | Whitford, Christopher M. | - |
dc.contributor.nonIdAuthor | Robertsen, Helene L. | - |
dc.contributor.nonIdAuthor | Blin, Kai | - |
dc.contributor.nonIdAuthor | Jorgensen, Tue S. | - |
dc.contributor.nonIdAuthor | Klitgaard, Andreas K. | - |
dc.contributor.nonIdAuthor | Gren, Tetiana | - |
dc.contributor.nonIdAuthor | Jiang, Xinglin | - |
dc.contributor.nonIdAuthor | Weber, Tilmann | - |
dc.description.isOpenAccess | Y | - |
dc.type.journalArticle | Article | - |
dc.subject.keywordAuthor | streptomycetes | - |
dc.subject.keywordAuthor | CRISPR base editor | - |
dc.subject.keywordAuthor | cytidine deaminase | - |
dc.subject.keywordAuthor | adenosine deaminase | - |
dc.subject.keywordAuthor | genome editing | - |
dc.subject.keywordPlus | COMPLETE GENOME SEQUENCE | - |
dc.subject.keywordPlus | DNA-REPAIR | - |
dc.subject.keywordPlus | RESISTANCE GENE | - |
dc.subject.keywordPlus | CLONING | - |
dc.subject.keywordPlus | EXPRESSION | - |
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