Highly Efficient Base Editing Systems In Streptomyces By Using Cas9-deaminase Fusions

The enormous interests and enthusiasm to the genus Streptomyces are aroused from a fact that it is one of the most important sources of pharmacologically active and industrially relevant secondary metabolites.Unfortunately,genetic manipulation of Streptomyces is much more difficult compared to other model organisms due to their filamentous growth,extremely high G+C content genomes and more diverse genomic contents,which has been a huge bottleneck for its functional investigation and metabolic engineering.Conventional CRISPR/Cas system has accelerated the genetic manipulation of these bacteria by introducing a double-strand break(DSB)to the genomic DNA,but in application this DSB dependent method was often arduous due to the low survival rate.In particular,current technology cannot easily deliver the desirable goal of multiple simultaneous precise editing in Streptomyces due to the inefficienct repair machinery.Therefore,more efficient and stable genome editing tools are highly needed.A newly-developed technique “base editing”,which derived from the CRISPR/Cas system,brings a new dawn for solving this conundrum.By fusing a single-stranded DNA-specific cytosine deaminase or an adenosine deaminase to an inactivated Cas protein,cytidine base editors(CBEs)and adenine base editors(ABEs)enable sg RNA targeted C-to-T or A-to-G nucleotide substitutions,respectively,without generating DSB and providing repair template.The advent and implementation of this system for use in eukaryotic genome editing has led to an explosion of advances in the life sciences over a remarkably short time.Motivated by it,we developed a set of efficient Streptomyces genome editing tools based on the BE technology.We achieved successful editing in Streptomyces at frequencies up to 100% by using nicked Cas9 guided base editors(BE3 and ABEn)and halved frequencies by using defective Cas9 guided base editors(BE2 and ABEd).Furthermore,we demonstrate the multiplex genome editing potential of BE3 by mutating nine target genes simultaneously.In addition,improved specificity is also determined by using the high-fidelity version of BE3(HF-BE3).This new thchnology no longer relies on the traditional homologous recombination and time-consuming target mutant screening.More importantly,for the first time it also provides a long-anticipated tool to inactivate multiple genes simultaneously.It is highly expected that our present results in this study will readily accelerate the in vivo functional analysis of biosynthetic pathways and the generation of secondary metabolite derivatives of Streptomyces and related Actinomycetes.