MAVERICC: Efficient marker-free rescue of vaccinia virus recombinants by in vitro CRISPR-Cas9 engineering

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, a highly 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 rapidly 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/425447v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@17151cborg.highwire.dtl.DTLVardef@b0f480org.highwire.dtl.DTLVardef@184166corg.highwire.dtl.DTLVardef@dc177d_HPS_FORMAT_FIGEXP M_FIG C_FIG Overview of MAVERICC. Conceptual overview of the approach outlined in this manuscript. To make VACV recombinants, the parental virus is first purified and vDNA is isolated with phenol:chloroform extraction. This purified vDNA is then treated with Cas9 enzyme and sgRNAs that are directed to a specific locus in the VACV genome. The cleaved vDNA is then transfected into FWPV-infected BSC-40 cells along with a transfer amplicon containing an insertion or mutation of interest flanked by homologous sequences. Recombination is allowed to occur for 5-7 days, during which time the cleaved vDNA is healed by the transfer amplicon, thus editing the VACV genome, and packaged into infectious viral particles. Individual plaques are grown up and rVACVs are isolated after a single round of plaque purification. Image was created with Biorender.com.