Developing oncolytic Herpes simplex virus type 1 through UL39 knockout by CRISPR-Cas9

Abstract

Objectives: Oncolytic Herpes simplex virus type 1 (HSV-1) has emerged as a promising strategy for cancer therapy. However, development of novel oncolytic mutants has remained a major challenge owing to low efficiency of conventional genome editing methods. Recently, CRISPR-Cas9 has revolutionized genome editing.

Materials and methods: In this study, we aimed to evaluate the capability of CRISPR-Cas9 to manipulate the UL39 gene to create oncolytic HSV-1. Herein, three sgRNAs were designed against the UL39 gene and transfected into HEK-293 cell line followed by infection with HSV-1 KOS.

Results: After three rounds of plaque purification, several HSV-1 mutants were identified by PCR analysis and sequencing. One of these mutations in which 55 nucleotides were deleted resulted in a frameshift mutation that in turn produced a truncated protein with only 167 amino acids from 1137 amino acids. Functional analysis in Vero and primary fibroblast cells revealed that viral replication was significantly lower and plaque size was smaller in the HSV-1 mutant compared with HSV-1 KOS. Moreover, the relative amount of viral genome present in the supernatants of infected cells (Vero and primary fibroblast cells) with HSV-1 mutant was significantly decreased compared with those of HSV-1 KOS.

Conclusion: Our data revealed that targeting UL39 with CRISPR-Cas9 could develop oncolytic HSV-1.

Keywords: CRISPR-Cas9; Herpes simplex virus type 1; Oncolytic virus; Ribonucleotide reductase; UL39.

Figure 1

Transfection efficiency of plasmids in HEK-293 cells

Figure 2

Genome PCR of isolated clones. Several clones (4, 9, 10, 11, 14, 16, 18, 20, 22, 27) were randomly isolated and target site of their UL39 gene was amplified via PCR and visualized via Electrophoresis. Each band represents a clone. The band of some clones locates lower than that of the wild type

Figure 3

Identification of CRISPR-Cas9 derived-indel mutations by restriction enzyme assay

Figure 4

Sequencing analysis of isolated clones

Figure 5

Schematic representation of RR protein in HSV-1 KOS (WT), HSV-1 mutant 2, HSV-1 mutant 18 and HSV-1 mutant 2- 1

Figure 6

HFF cell cultures infected with the HSV-1 mutants 18, 2 and HSV-1 KOS (WT). Control non infected cell cultures (A) and cytopathic effect HSV-1 KOS (WT) at 72h (B), cytopathic effect HSV-1 mutants18 at 72h (C) and cytopathic effect HSV-1 mutants 2 at 72 hr (D) post-infection. Unstained fresh cultures (40X))

Figure 7

Vero cell cultures infected with the HSV-1 mutants 18, 2 and HSV-1 KOS (WT). Control non infected cell cultures (A) and cytopathic effect HSV-1 KOS (WT) at 72 hr (B), cytopathic effect HSV-1 mutants 2 at 72 hr (C) and cytopathic effect HSV-1 mutants18 at 72h (D) post-infection. Unstained fresh cultures (40X))

Figure 8

Viral growth kinetics in HFF (A) and Vero (B) cells. Growth kinetics of HSV-1 KOS (WT), HSV-1 mutant 18 and HSV-1 mutant 2 in HFF and Vero cells were assessed. When confluency reached 90%, the cells were infected with viruses at a multiplicity of infection of 0.01. Supernatants were collected at 24, 48, and 72 hr post infection and consequently the viral titers were determined by plaque assays on Vero cells

Figure 9

Relative amount of viral DNA in HFF (A) and Vero (B) cells

Figure 10

Plaque features of HSV-1 mutants18, 2 and HSV-1 KOS (WT). Plaques of HSV-1 mutant 18 were significantly smaller than those of HSV-1 mutant 2 and HSV-1 KOS (WT). Vero cells were infected with HSV-1 KOS (WT) and HSV-1 mutants 18, 2 then overlaid with 1.5% agarose. 3 days post infection, plaques were fixed with formaldehyde10 % and stained with 1% crystal violet solution