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

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

169 Downloads (Pure)


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.

Original languageEnglish
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number41
Pages (from-to)20366-20375
Publication statusPublished - 2019


  • Adenosine deaminase
  • CRISPR base editor
  • Cytidine deaminase
  • Genome editing
  • Streptomycetes

Fingerprint Dive into the research topics of 'Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST'. Together they form a unique fingerprint.

Cite this