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

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Abstract

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-ULstr 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-ULstr, 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-ULstr 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-ULstr could be employed for efficient multiplex genome editing in Streptomyces. Since the dCas9-CDA-ULstr-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.

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Acknowledgements

This work was supported by the National Drug Innovation Major Project (2018ZX09711001-006-012), the National Natural Science Foundation of China (31770088, 31570072 and 31430004), the Science and Technology Commission of Shanghai Municipality (18ZR1446700), the Derivative Bank of Chinese Biological Resources, CAS (ZSYS-016), and Shanghai Engineering Research Center of Plant Germplasm Resources (17DZ2252700).

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Authors and Affiliations

  1. College of Life Sciences, Shanghai Normal University, Shanghai, 200234, China

    Yawei Zhao, Jun Chen, Chuanwen Sun & Yinhua Lu

  2. State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200030, China

    Yawei Zhao, Zixin Deng & Zhijun Wang

  3. Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China

    **zhong Tian, Guosong Zheng & Weihong Jiang

  4. School of Life Science, Taizhou University, Taizhou, 318000, China

    Zhongyi Yang

  5. Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, 142290, Russia

    Andrei A. Zimin

  6. Shanghai Engineering Research Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai, 200234, China

    Yinhua Lu

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  1. Yawei Zhao
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Zhao, Y., Tian, J., Zheng, G. et al. Multiplex genome editing using a dCas9-cytidine deaminase fusion in Streptomyces. Sci. China Life Sci. 63, 1053–1062 (2020). https://doi.org/10.1007/s11427-019-1559-y

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