Highly Efficient Base Editing in Viral Genome Based on Bacterial Artificial Chromosome Using a Cas9-Cytidine Deaminase Fused Protein
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.
KeywordsPseudorabies virus (PRV) Bacterial artificial chromosome (BAC) Base-editing CRISPR/Cas9 Genome editing
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.
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.
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