Figure 6. Effect of CRISPR-Cas9 on input HSV genomes.
(A) HFFs transduced with lentivirus expressing Cas9 and sgRNA were infected with HSV-1 at an MOI of 1 in the presence or absence of PAA and harvested at 10 hpi. Proteins were detected by immunoblotting with antibodies specific for the indicated proteins. Immunoblots of GAPDH are shown as a control. (B and C) HFFs transduced with lentivirus expressing SaCas9 and sgRNA were infected with HSV-1 at an MOI of 3 in the presence of PAA and harvested at the indicated times post infection. The accumulated DNAs were detected by real time qPCR amplifying within the UL29 gene (B) or over the UL30-5 sgRNA (C) target site. The histogram shows the mean values and standard deviations from biological replicates (N ≥ 3, Ratio paired t test, **p<0.01 and ***p<0.001). (D and E) PCR amplification across the UL30-5 target site in quiescent d109 genomes and replicating HSV-1 genomes. Quiescently infected HFF cells were transduced with lentivirus expressing Cas9/UL30-5 sgRNA as described in Figure 2A and analyzed at the UL30-5 sgRNA target site by qPCR. The qPCR across the Cas9/UL30-5 sgRNA target site (UL30-5 PCR) was normalized to GAPDH (D) or a remote site of qPCR (E, UL29 PCR) with (UL30-5) or without (Cas9) UL30-5 sgRNA expression. As a control, Cas9 ± UL30-5 sgRNA transduced HFF cells were infected with HSV-1, harvested at 12 hpi and analyzed as described above. Cas9: no sgRNA, UL30-5: Cas9 with UL30-5 sgRNA. The histogram shows the mean values and standard deviations from biological replicates of d109 (N = 6) or HSV-1 (N = 3) infected cells (t-test: ****p<0.0001).