Selection of Vaccinia Virus Recombinants Using CRISPR/Cas9

Date

2021

Authors

Gowripalan, Anjali
Smith, Stewart
Tscharke, David

Journal Title

Journal ISSN

Volume Title

Publisher

Bio-Protocol

Abstract

The engineering of poxvirus genomes is fundamental to primary and applied virology research. Indeed, recombinant poxviruses form the basis for many novel vaccines and virotherapies but producing and purifying these viruses can be arduous. In recent years, CRISPR/Cas9 has become the favoured approach for genome manipulation due to its speed and high success rate. However, recent data suggests poxvirus genomes are not repaired well following Cas9 cleavage. As a result, CRISPR/Cas9 is inefficient as an editing tool, but very effective as a programmable selection agent. Here, we describe protocols for the generation and enrichment of recombinant vaccinia viruses using targeted Cas9 as a selection tool. This novel use of Cas9 is a simple addition to current homologous recombination-based methods that are widespread in the field, facilitating implementation in laboratories already working with poxviruses. This is also the first method that allows for isolation of new vaccinia viruses in less than a fortnight, without the need to incorporate a marker gene or manipulation of large poxvirus genomes in vitro and reactivation with helper viruses. Whilst this protocol describes applications for laboratory strains of vaccinia virus, it should be readily adaptable to other poxviruses.

Description

Keywords

Recombinant virus, Vaccinia virus, VACV, CRISPR/Cas9 selection, Virotherapy, Vaccine development

Citation

Source

Bio-Protocol

Type

Journal article

Book Title

Entity type

Access Statement

Open Access

License Rights

DOI

10.21769/BioProtoc.4270

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