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. 2016 Mar 7;8(3):72. doi: 10.3390/v8030072

Figure 1.

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.