The application of CRISPR/Cas9 to improve genome engineering efficiency of large dsDNA viruses has been extensively described, but a robust and versatile method for high-throughput generation of marker-free recombinants for a desire locus has not been reported yet. Cytoplasmic-replicating viruses use their own repair enzymes for homologous recombination, while nuclear-replicating viruses use the host repair machinery. This is translated into a wide range of Cas9-induced homologous recombination efficiency depending on the virus replication compartment and viral/host repair machinery characteristics and accessibility. However, the use of Cas9 as a selection agent to target parental virus genomes robustly improves the selection of desired recombinants across large dsDNA viruses. We used ectromelia virus (ECTV) and herpes simplex viruses (HSV) type 1 and 2, to optimize a CRISPR/Cas9 method that can be versatilely used for efficient genome editing and selection of both cytoplasmic- and nuclear-replicating viruses. We performed a genome-wide genetic variant analysis of mutations located at predicted off-target sequences for 20 different recombinants, showing off-target-free accuracy by deep-sequencing. Our results support this optimized method as an efficient, accurate and versatile approach to enhance the two critical factors of high-throughput viral genome engineering: generation and color-based selection of recombinants. This application of CRISPR/Cas9 reduces time and labor of screening of desired recombinants, allowing for high-throughput generation of large collections of mutant dsDNA viruses for a desire locus in less than two weeks.

This work was funded by the Spanish Ministry of Science and Innovation and European Union (European Regional Development’s Funds, FEDER) (grants SAF2015-67485-R and RTI2018-097581-B100), and a PhD studentship from Ministerio de Educación, Cultura y Deporte awarded to ADLM (FPU13/05425).

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