CRISPR-Cas9 as a Powerful Tool for Efficient Creation of Oncolytic Viruses - PubMed (original) (raw)
Review
CRISPR-Cas9 as a Powerful Tool for Efficient Creation of Oncolytic Viruses
Ming Yuan et al. Viruses. 2016.
Abstract
The development of oncolytic viruses has led to an emerging new class of cancer therapeutics. Although the safety profile has been encouraging, the transition of oncolytic viruses to the clinical setting has been a slow process due to modifications. Therefore, a new generation of more potent oncolytic viruses needs to be exploited, following our better understanding of the complex interactions between the tumor, its microenvironment, the virus, and the host immune response. The conventional method for creation of tumor-targeted oncolytic viruses is based on homologous recombination. However, the creation of new mutant oncolytic viruses with large genomes remains a challenge due to the multi-step process and low efficiency of homologous recombination. The CRISPR-associated endonuclease Cas9 has hugely advanced the potential to edit the genomes of various organisms due to the ability of Cas9 to target a specific genomic site by a single guide RNA. In this review, we discuss the CRISPR-Cas9 system as an efficient viral editing method for the creation of new oncolytic viruses, as well as its potential future applications in the development of oncolytic viruses. Further, this review discusses the potential of off-target effects as well as CRISPR-Cas9 as a tool for basic research into viral biology.
Keywords: CRISPR-Cas9; Herpes simplex virus; Oncolytic virus; Vaccinia virus; adenovirus; homologous recombination.
Figures
Figure 1
Construction of gRNA vector and repair donor vector for creation of a mutant vaccinia virus. (A) A 19mers or 20mers gRNA target sequence with/without a G at 5′ end is designed within the target gene; (B) DNA sequence of a gRNA target region is cloned into a gRNA vector using U6 at the promoter; and (C) a repair donor vector is constructed. The length of right arm and left arm is about 500 bp, both arms can just flank the target gene, or slightly overlap with target gene up to 50 bp. The purification marker RFP driven by the vaccinia virus promoter H5 is cloned between the right arm and left arm in the donor vector, a therapeutic gene driven by H5 promoter, such as human granulocyte-macrophage colony-stimulating factor (hGM-CSF), can be cloned into the site between RFP and the left arm. The RFP or a therapeutic gene does not need a poly A signal to stabilize the mRNA as the mRNA is transcribed in the cytoplasm.
Figure 2
gRNA guided Cas9 induces homologous recombination by creating a DNA double-stranded break in the target region of vaccinia virus. (A) The gRNA is transcribed; (B) gRNA guides Cas9 to the target site; (C) Cas9 creates a DNA double-stranded break, which is repaired by the repair donor vector via the mechanism of homologous recombination; and (D) the mutant vaccinia virus is generated with the deletion of the target gene and insertion of the purification marker RFP containing its promoter H5. A therapeutic gene hGM-CSF and its promoter H5 can also be incorporated into the target gene.
Figure 2
gRNA guided Cas9 induces homologous recombination by creating a DNA double-stranded break in the target region of vaccinia virus. (A) The gRNA is transcribed; (B) gRNA guides Cas9 to the target site; (C) Cas9 creates a DNA double-stranded break, which is repaired by the repair donor vector via the mechanism of homologous recombination; and (D) the mutant vaccinia virus is generated with the deletion of the target gene and insertion of the purification marker RFP containing its promoter H5. A therapeutic gene hGM-CSF and its promoter H5 can also be incorporated into the target gene.
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