Multiplex genome editing using a dCas9-cytidine deaminase fusion in Streptomyces

CRISPR/Cas-mediated genome editing has greatly facilitated the study of gene function in Streptomyces. However, it could not be efficiently employed in streptomycetes with low homologous recombination (HR) ability. Here, a deaminase-assisted base editor dCas9-CDA-UL_str was developed in Streptomyces, which comprises the nuclease-deficient Cas9 (dCas9), the cytidine deaminase from Petromyzon marinus (PmCDA1), the uracil DNA glycosylase inhibitor (UGI) and the protein degradation tag (LVA tag). Using dCas9-CDA-UL_str, we achieved single-, double- and triple-point mutations (cytosine-to-thymine substitutions) at target sites in Streptomyces coelicolor with efficiency up to 100%, 60% and 20%, respectively. This base editor was also demonstrated to be highly efficient for base editing in the industrial strain, Streptomyces rapamycinicus, which produces the immunosuppressive agent rapamycin. Compared with base editors derived from the cytidine deaminase rAPOBEC1, the PmCDA1-assisted base editor dCas9-CDA-UL_str could edit cytosines preceded by guanosines with high efficiency, which is a great advantage for editing Streptomyces genomes (with high GC content). Collectively, the base editor dCas9-CDA-UL_str could be employed for efficient multiplex genome editing in Streptomyces. Since the dCas9-CDA-UL_str-based genome editing is independent of HR-mediated DNA repair, we believe this technology will greatly facilitate functional genome research and metabolic engineering in Streptomyces strains with weak HR ability.