Harnessing stepping-stone hosts to engineer, select, and reboot synthetic bacteriophages in one pot

Li Cheng; Ziqing Deng; Haoran Tao; Wenchen Song; Bo Xing; Wenfeng Liu; Lingxin Kong; Shengjian Yuan; Yingfei Ma; Yayun Wu; Xun Huang; Yun Peng; Nai-Kei Wong; Yingxia Liu; Yun Wang; Yue Shen; Junhua Li; Minfeng Xiao

doi:10.1016/j.crmeth.2022.100217

Harnessing stepping-stone hosts to engineer, select, and reboot synthetic bacteriophages in one pot

Cell Rep Methods. 2022 May 23;2(5):100217. doi: 10.1016/j.crmeth.2022.100217.

Authors

Li Cheng^{1

2

3}, Ziqing Deng^{1

3

4}, Haoran Tao^{1

5

3}, Wenchen Song^{1

3}, Bo Xing^{1

5

3}, Wenfeng Liu^{1

3}, Lingxin Kong^{1

5

3}, Shengjian Yuan⁶, Yingfei Ma⁶, Yayun Wu⁷, Xun Huang⁷, Yun Peng⁸, Nai-Kei Wong⁸, Yingxia Liu⁸, Yun Wang^{1

9

10}, Yue Shen^{1

9

11}, Junhua Li^{1

2

3}, Minfeng Xiao^{1

3}

Affiliations

¹ BGI-Shenzhen, Shenzhen 518083, China.
² School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China.
³ Shenzhen Key Laboratory of Unknown Pathogen Identification, BGI-Shenzhen, Shenzhen 518083, China.
⁴ BGI-Beijing, Beijing 100101, China.
⁵ University of the Chinese Academy of Sciences, Beijing 101408, China.
⁶ Shenzhen Key Laboratory of Synthetic Genomics, Guangdong Provincial Key Laboratory of Synthetic Genomics, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
⁷ Infection Control Center, Xiangya Hospital, Central South University, Changsha 410008, Hunan, China.
⁸ Shenzhen Key Laboratory of Pathogen and Immunity, Guangdong Key Laboratory for Diagnosis and Treatment of Emerging Infectious Diseases, State Key Discipline of Infectious Disease, Second Hospital Affiliated to Southern University of Science and Technology, Shenzhen Third People's Hospital, 518112 Shenzhen, China.
⁹ Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, Shenzhen 518120, China.
¹⁰ Guangdong Provincial Academician Workstation of BGI Synthetic Genomics, BGI-Shenzhen, Shenzhen 518120, China.
¹¹ Shenzhen Engineering Laboratory for Innovative Molecular Diagnostics, BGI-Shenzhen, Shenzhen 518120, China.

Abstract

Advances in synthetic genomics have led to a great demand for genetic manipulation. Trimming any process to simplify and accelerate streamlining of genetic code into life holds great promise for synthesizing and studying organisms. Here, we develop a simple but powerful stepping-stone strategy to promote genome refactoring of viruses in one pot, validated by successful cross-genus and cross-order rebooting of 90 phages infecting 4 orders of popular pathogens. Genomic sequencing suggests that rebooting outcome is associated with gene number and DNA polymerase availability within phage genomes. We integrate recombineering, screening, and rebooting processes in one pot and demonstrate genome assembly and genome editing of phages by stepping-stone hosts in an efficient and economic manner. Under this framework, in vitro assembly, yeast-based assembly, or genetic manipulation of native hosts are not required. As additional stepping-stone hosts are being developed, this framework will open doors for synthetic phages targeting more pathogens and commensals.

Keywords: antibiotic resistance; intermediate host; in vivo genome assembly; phage biology; phage engineering; phage therapy; synthetic biology; synthetic genomics.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacteriophages* / genetics
Base Sequence
DNA-Directed DNA Polymerase / genetics
Gene Editing
Genomics

Substances

DNA-Directed DNA Polymerase