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

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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.
Publisher
NATL ACAD SCIENCES
Issue Date
2019-10
Language
English
Article Type
Article
Citation

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, v.116, no.41, pp.20366 - 20375

ISSN
0027-8424
DOI
10.1073/pnas.1913493116
URI
http://hdl.handle.net/10203/268088
Appears in Collection
CBE-Journal Papers(저널논문)
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