Recombinant Virus Construction via CRISPR

Based on the methods of Bi et al. [23 (link)], the recombinant virus with a partial UL7 deletion mutation was constructed via co-transfection of plasmids PX330-UL7-1 and PX330-UL7-2. Virus was harvested from infected 293 T cells at 48 h post-infection (p.i.), and viral genomic DNA was extracted using the TIANamp Virus RNA/DNA Kit (Tiangen, Beijing, China). The genomic region surrounding the CRISPR target site of the UL7 gene was PCR-amplified using PrimeSTAR DNA polymerase (TAKARA, Dalian, Liaoning, China) with the primers UL7-sense and UL7-antisense. The PCR products were purified using the Universal DNA Purification Kit (Tiangen, Beijing, China). Purified PCR products (400 ng) amplified from the genomic DNA extraction were re-annealed and treated with SURVEYOR nuclease (Transgenomics, Omaha, NE, U.S.A.). A nuclease capable of recognizing and digesting the previously mismatched nucleotides in the genome was used to confirm that the mutation in the UL7 gene was created by the CRISPR approach. The products were analyzed on 10 % TBE polyacrylamide gels, which were stained with ethidium bromide (EB) and imaged using a Bio-Rad Gel Doc gel imaging system. Quantification was based on the relative band intensities, as described by Cong et al. [24 (link)]. After detecting the mutation efficiency, mutated viruses were purified via plaque assay.