Highly efficient DSB free base editing for streptomycetes with CRISPR BEST
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. 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 protospacer identification process, we also updated the CRISPy web (https:/crispy.secondarymetabolites.org) and now it allows designing sgRNAs specifically for CRISPR BEST applications.
Proceedings of the National Academy of Sciences