CRISPR/Cas9 mediated gene editing is a good alternative to BACs for GPCMV and the use of CRISPR/Cas9 will be demonstrated in this paper. A gene, GP133, was chosen to test if CRISPR/Cas9 could target GPCMV and if Cas9 would be harmful to the replication process. The GP133 was cloned and used to create a guide RNA (gRNA) which would be attached to the Cas9 for the CRISPR process and a nontargeting gRNA would be used as well as a control. GPL cells were transfected with gRNA plasmids then transfected with an expression construct which transfected half of the cells. They were then treated with puromycin after 48 hours. After 24 hours of treatment, the cells were infected with RFP GPCMV. This caused cell death in many of the cells. Multiplicity of infection (MOI) of one in untreated cells was used. DNA was then taken from infected cells and were either subjected to cleavage detection assay or PvuI digestion. Cleavage detection assay was done by allowing the formation of Heteroduplexes. These Heteroduplexes are predicted to cleave fragments at 441 and 141 bp and show that mutants are being generated by Cas9 and there is repair by NHEJ. PvuI digestion shows that NHEJ does not repair, but instead destroys PvuI digestion site. The DNA was all mutated at the Cas9 cleavage site. GP133 was then modified through deletion and insertion to see how it would react. CRISPR/Cas9 generate gene disrupting indel mutations.

A)CRISPR site in GP133 was looked at for the initial experiments.Cas9 Cleavage site and PvuI overlap. PvuI is a restriction site. Restriction sites are specific sequences of nucleotides which can then be targeted by a restriction enzyme to be cut. B)GP133 gRNA was created along with a nontargeting (NT) gRNA which is used to compare how replication is affected in the absence of a specific target. A ~582 bp DNA fragment was amplified with infected cell DNA. (-) PCR amplified using infected cells. (+) Cleavage detection assay (No CDA in NT while GP133 was). (P) PvuI Restriction (NT is cleaved by PvuI but GP133 repairs itself after cleavage and destroys the PvuI site, which leaves a full-length PCR product) The repair and cleavage of GP133 occurred and there were mutations of all the viral DNA. C) Titers of both were collected to see if Cas9 was harmful to viral replication. Across 2 experiments there was a decrease in the recovery of virus from cells transfected with GP133 gRNA plasmids while the NT gRNA had a higher virus recovery. This shows that Cas9 has little influence on viral replication. D) GPCMV mutants were isolated by limiting dilution from virus containing media from the GP133 gRNA transfection/infection. DNA is then extracted from the 15 isolated plaques and PCR amplified and sanger sequenced. All 15 viruses had mutations, 11 of which are unique. 10/11 had deletions on the Cas9 cleavage site. 9 had frameshifts. Only 2 remained in frame