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.