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Significance 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.

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