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Highly Efficient Base Editing in Viral Genome Based on Bacterial Artificial Chromosome Using a Cas9-Cytidine Deaminase Fused Protein

  • Ke Zheng
  • Fang-Fang Jiang
  • Le Su
  • Xin Wang
  • Yu-Xin Chen
  • Huan-Chun Chen
  • Zheng-Fei LiuEmail author
RESEARCH ARTICLE

Abstract

Viruses evolve rapidly and continuously threaten animal health and economy, posing a great demand for rapid and efficient genome editing technologies to study virulence mechanism and develop effective vaccine. We present a highly efficient viral genome manipulation method using CRISPR-guided cytidine deaminase. We cloned pseudorabies virus genome into bacterial artificial chromosome, and used CRISPR-guided cytidine deaminase to directly convert cytidine (C) to uridine (U) to induce premature stop mutagenesis in viral genes. The editing efficiencies were 100%. Comprehensive bioinformatic analysis revealed that a large number of editable sites exist in pseudorabies virus (PRV) genomes. Notably, in our study viral genome exists as a plasmid in E. coli, suggesting that this method is virus species-independent. This application of base-editing provided an alternative approach to generate mutant virus and might accelerate study on virulence and vaccine development.

Keywords

Pseudorabies virus (PRV) Bacterial artificial chromosome (BAC) Base-editing CRISPR/Cas9 Genome editing 

Notes

Acknowledgements

This work was supported by the National Key Research and Development Program (2016YFD0500105) and the Natural Science Foundation of China (31770191). We thank Professor Mei-Lin Jin for providing Cre expression plasmid.

Author Contributions

ZFL and KZ designed the experiments; KZ performed genome editing in PRV BAC; XW and YXC constructed the PRV BAC; FFJ and LS investigated characteristic of PRV mutant; HCC provided PRV; KZ, and ZFL wrote and revised the manuscript.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Animal and Human Rights Statement

This article does not contain any studies with human or animal subjects performed by any of the authors.

Supplementary material

12250_2019_175_MOESM1_ESM.pdf (233 kb)
Figure S1 iSTOP mutation stability of PRV-TgE within 10 passages. PRV-TgE mutant was sub-cultured for ten passages, one microliter of cell lysate was taken as template for PCR amplification and Sanger sequencing. The substituted bases were marked with red arrows. (PDF 233 kb)
12250_2019_175_MOESM2_ESM.xlsx (10 kb)
Table S1. Sequences of oligonucleotides used in this study (XLSX 11 kb)
12250_2019_175_MOESM3_ESM.xlsx (83 kb)
Table S2. The editable iSTOP codons information in PRV-Ea genome (XLSX 84 kb)
12250_2019_175_MOESM4_ESM.xlsx (10 kb)
Table S3. The count of editable iSTOP codons in PRV-Ea genome (XLSX 10 kb)
12250_2019_175_MOESM5_ESM.xlsx (267 kb)
Table S4. The editable iSTOP codons information in PRV-Becker genome (XLSX 267 kb)
12250_2019_175_MOESM6_ESM.xlsx (277 kb)
Table S5. The editable iSTOP codons information in PRV-HNX genome (XLSX 277 kb)

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Copyright information

© Wuhan Institute of Virology, CAS 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Agricultural Microbiology and Key Laboratory of Preventive Veterinary Medicine in Hubei Province, College of Veterinary MedicineHuazhong Agricultural UniversityWuhanChina
  2. 2.Gene Editing Research CenterHebei University of Science and TechnologyShijiazhuangChina

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