MAVERICC: Marker-free Vaccinia Virus Engineering of Recombinants through in vitro CRISPR/Cas9 Cleavage

Ethan Laudermilch, Kartik Chandran

Research output: Contribution to journalArticlepeer-review

Abstract

Vaccinia virus (VACV)-based vectors are in extensive use as vaccines and cancer immunotherapies. VACV engineering has traditionally relied on homologous recombination between a parental viral genome and a transgene-bearing transfer plasmid, an inefficient process that necessitates the use of a selection or screening marker to isolate recombinants. Recent extensions of this approach have sought to enhance the recovery of transgene-bearing viruses through the use of CRISPR-Cas9 engineering to cleave the viral genome in infected cells. However, these methods do not completely eliminate the generation of WT viral progeny and thus continue to require multiple rounds of viral propagation and plaque purification. Here, we describe MAVERICC (marker-free vaccinia virus engineering of recombinants through in vitro CRISPR/Cas9 cleavage), a new strategy to engineer recombinant VACVs in a manner that overcomes current limitations. MAVERICC also leverages the CRISPR/Cas9 system but requires no markers and yields essentially pure preparations of the desired recombinants in a single step. We used this approach to introduce point mutations, insertions, and deletions at multiple locations in the VACV genome, both singly and in combination. The efficiency and versatility of MAVERICC make it an ideal choice for generating mutants and mutant libraries at arbitrarily selected locations in the viral genome to build complex VACV vectors, effect vector improvements, and facilitate the study of poxvirus biology.

Original languageEnglish (US)
Article number166896
JournalJournal of Molecular Biology
Volume433
Issue number9
DOIs
StatePublished - Apr 30 2021

Keywords

  • CRISPR/Cas9
  • Helper virus
  • Poxvirus
  • Recombinant vaccinia viruses
  • Viral vector

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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