CRISPR-Cas9 Expressed in Stably Transduced Cell Lines Promotes Recombination and Selects for Herpes Simplex Virus Recombinants

Abstract

Recombinant herpes simplex virus strains can be constructed by several methods, including homologous recombination, bacterial artificial chromosome manipulation, and yeast genetic methods. Homologous recombination may have the advantage of introducing fewer genetic alterations in the viral genome, but the low level of recombinants can make this method more time consuming if there is no screen or selection. In this study we used complementing cell lines that express Cas9 and guide RNAs targeting the parental virus to rapidly generate recombinant viruses. Analysis of the progeny viruses indicated that CRISPR-Cas9 both promoted recombination to increase recombinant viruses and selected against parental viruses in the transfection progeny viruses. This approach can also be used to enrich for recombinants made by any of the current methods.

Figure 1.. Schematic diagram of the gRNA target sites in the plasmid shuttle vector.

(A) Diagrams of d106S genome and pd27B shuttle vector. The clear boxes show the repeated sequences and the red lines indicate deleted sites of ICP22, ICP27, and two ICP4. The deleted ICP27 (UL54) location is represented in two angled lines. The expended map represents details of organization of sequences including CMV promoter (CMVp, light red), GFP (green), poly-A tail (poly-A, light blue), and HSV-1 sequences of UL54 and UL53 (gray). GFP gene can be replaced with Gene of Interest (GOI, in this study, mCherry, EBOV-GP, or SARS-CoV-2 spike) by homologous recombination with the shuttle vector of pd27B. (B) Sequences shown are the original sequence in pd27B plasmid (top) and synthesized gBlock sequence (bottom). The targeting regions for three gRNAs (gRNA-1, −2, and −3) in the pd27B shuttle plasmid are indicated by the blue arrow lines. Homologous sequences are shown in black bars and nucleic acids define the differences between pd27B and gBlock (also pd27Bmut). (C) Maps of the original shuttle plasmid (pd27B) and mutated shuttle plasmid (pd27Bmut). pd27B has flanking homologous sequences (UL54 and UL53) to HSV-1 DNA, CMVp (light red), GOI (light brown), and poly-A (light blue). Alterations in the three gRNA sequences located near the 5’ end of the UL53 gene were introduced into pd27mut using the gBlock (yellow). Co-transfection of pd27Bmut and d106S genome to complementing cell line E11 produces GOI inserted d106S virus.

Figure 2.. Effects of CRISPR/Cas9 on HSV-1 replication in different gRNA cell lines.

(A) Expression of Cas9 in the various cell lines. E11 cells transduced with lentiviruses expressing SaCas9 and sgRNA were harvested to yield lysates, and Cas9-FLAG was detected in the lysates by immunoblotting using antibodies specific for FLAG. Immunoblots of GAPDH are shown as a loading control. (B) Replication of d106S virus in the Cas9/gRNA cell lines. Parental E11 and gRNA/Cas9 expressing E11 cells (#1, #2, #3, and #1+2+3) were infected with wildtype HSV-1 (MOI of 0.1 or 0.01), harvested at 48 hpi, and viral titers were determined by plaque assay. -gRNA: Cells expressing Cas9 but no gRNA. The graph shows the mean values and standard errors from three biological replicate experiments and log-transformed data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Statistical significance relative to -gRNA control is presented. * p<0.05, *** p<0.001, and **** p<0.0001.

Figure 3.. Effect of CRISPR/Cas9 on Viral Progeny from Co-Transfected Cultures.

Viral d106S DNA and linearized shuttle plasmid were co-transfected and harvested at 7 dpi. GFP-positive (parental d106S) plaques and GPF-negative (recombinant virus, either mCherry or EBOV-GP) plaques in the harvested virus were counted in serial dilutions of the progeny virus from the various transfection progeny. (A) The numbers of GFP-positive and -negative plaques in the various transfection progeny are shown. (B) The percentages of GFP-negative viruses were calculated for each sample. Cells #1, #2, #3, and #1+2+3 indicate gRNA-expressing cell lines that were used for co-transfection of d106S and a shuttle plasmid. -gRNA: Cells with Cas9 but no gRNA. The graph shows the mean values and standard errors from combined two biological replicate experiments with one-way ANOVA with Tukey’s multiple comparisons test, ** p<0.01 and *** p<0.001.

Figure 4.. Verification of the integrity and expression of the transgene in d106S recombinants.

(A) Verification of transgene in the HSV-1 d106S recombinants. E11 cells were infected with recombinant d106S-CoV2-S or the d106S vector at an MOI of 10. Total DNA from the infections was isolated, and PCR was carried out on the DNA with primers specific for SARS-CoV2 spike gene within the recombinant virus. (B) Immunoblotting for SARS-CoV-2 virus spike protein in infected cell lysates. Human foreskin fibroblast (HFF) cells were infected with each purified clone of the d106S-CoV2-S recombinants. Infected cells were harvested at 24 hpi. Proteins in the cell lysate were separated by SDS-PAGE and probed with a rabbit antibody for the SARS-CoV-2 spike protein as the primary antibody. Antibody for the GAPDH protein was used as a loading control.