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Highly Efficient DSB-free Base Editing for Streptomycetes With CRISPR-BEST

Highly Efficient DSB-free Base Editing for Streptomycetes With CRISPR-BEST

Yaojun Tong, Christopher M Whitford, Helene L Robertsen, Kai Blin, Tue S Jørgensen, Andreas K Klitgaard, Tetiana Gren, Xinglin Jiang, Tilmann Weber, Sang Yup Lee

Proc Natl Acad Sci U S A. 2019 Oct 8;116(41):20366-20375.

PMID: 31548381

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 Tü365, 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 protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP50935712	Kirromycin from Streptomyces collinus		50935-71-2	Price

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