1887

Journal of General Virology header logo

Volume 103, Issue 10

High-throughput engineering of cytoplasmic- and nuclear-replicating large dsDNA viruses by CRISPR/Cas9 Open Access

Abstract

The application of CRISPR/Cas9 to improve genome engineering efficiency for large dsDNA viruses has been extensively described, but a robust and versatile method for high-throughput generation of marker-free recombinants for a desired locus has not yet been reported. 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 efficiencies, 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 virus (HSV) type 1 and 2 to optimize a CRISPR/Cas9 method that can be used versatilely 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 colour-based selection of recombinants. This application of CRISPR/Cas9 reduces the time and labour for screening of desired recombinants, allowing for high-throughput generation of large collections of mutant dsDNA viruses for a desired locus, optimally in less than 2 weeks.

  • Received:
  • Accepted:
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Keyword(s): CRISPR/Cas9 , dsDNA viruses , genome engineering , off-target-free and two progenies
Funding
This study was supported by the:
  • Ministerio de Educación y Formación Profesional (Award FPU13/05425)
    • Principle Award Recipient: AlbertoDomingo López-Muñoz
  • European Regional Development Fund (Award RTI2018-097581-B100)
    • Principle Award Recipient: AntonioAlcami
  • Ministerio de Ciencia e Innovación (Award SAF2015-67485-R)
    • Principle Award Recipient: AntonioAlcami
© 2022 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2022-10-19
2024-05-04
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High-throughput engineering of cytoplasmic- and nuclear-replicating large dsDNA viruses by CRISPR/Cas9
J. Gen. Virol. 103, 001797 (2022); https://doi.org/10.1099/jgv.0.001797
/content/journal/jgv/10.1099/jgv.0.001797
/content/journal/jgv/10.1099/jgv.0.001797
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