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

• Published in: Science China. Life sciences 2020-07, Vol.63 (7), p.1053-1062
• Main Authors: Zhao, Yawei, Tian, Jinzhong, Zheng, Guosong, Chen, Jun, Sun, Chuanwen, Yang, Zhongyi, Zimin, Andrei A., Jiang, Weihong, Deng, Zixin, Wang, Zhijun, Lu, Yinhua
• Format: Article
• Language: English
• Subjects: Biomedical and Life Sciences; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Cas Systems - genetics; Cytidine deaminase; Cytidine Deaminase - genetics; Cytosine; Deoxyribonucleic acid; DNA; DNA glycosylase; DNA repair; Editors; Gene Editing - methods; Gene Expression Regulation, Bacterial; Genetic Engineering; Genome editing; Genome, Bacterial - genetics; Genomes; Guanosine - metabolism; Homologous recombination; Immunosuppressive Agents - metabolism; Life Sciences; Metabolic engineering; Nuclease; Point Mutation - genetics; Promoter Regions, Genetic; Rapamycin; Recombinant Proteins - genetics; Research Paper; Sirolimus - metabolism; Streptomyces; Streptomyces - genetics; Thymine; Uracil; Uracil-DNA glycosidase
• ISSN: 1674-7305; 1869-1889
• DOI: 10.1007/s11427-019-1559-y

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.