<P><B>Significance</B></P><P>Although CRISPR-Cas9 tools dramatically simplified the genetic manipulation of actinomycetes, significant concerns of genome instability caused by the DNA double-strand breaks (DSBs) and commo...
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https://www.riss.kr/link?id=A107460400
2019
-
SCOPUS,SCIE
학술저널
20366-20375(10쪽)
0
상세조회0
다운로드다국어 초록 (Multilingual Abstract)
<P><B>Significance</B></P><P>Although CRISPR-Cas9 tools dramatically simplified the genetic manipulation of actinomycetes, significant concerns of genome instability caused by the DNA double-strand breaks (DSBs) and commo...
<P><B>Significance</B></P><P>Although CRISPR-Cas9 tools dramatically simplified the genetic manipulation of actinomycetes, significant concerns of genome instability caused by the DNA double-strand breaks (DSBs) and common off-target effects remain. To address these concerns, we developed CRISPR-BEST, a DSB-free and high-fidelity single-nucleotide–resolution base editing system for streptomycetes and validated its use by determining editing properties and genome-wide off-target effects. Furthermore, our CRISPR-BEST toolkit supports Csy4-based multiplexing to target multiple genes of interest in parallel. We believe that our CRISPR-BEST approach is a significant improvement over existing genetic manipulation methods to engineer streptomycetes, especially for those strains that cannot be genome-edited using normal DSB-based genome editing systems, such as CRISPR-Cas9.</P><P>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 <I>Streptomyces</I> species. Particularly in nonmodel actinomycete <I>Streptomyces collinus</I> Tu¨365, CRISPR-cBEST efficiently inactivated the 2 copies of <I>kirN</I> 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 protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.</P>