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. 2019 Dec 2;8:e51662. doi: 10.7554/eLife.51662

Figure 8. Effect of CRISPR-Cas9 on non-coding and non-essential regions of the HSV-1 genome.

(A and B) HFFs transduced with lentivirus expressing SaCas9 and sgRNAs were infected with HSV-1 at an MOI of 0.1 (A) or 5 (B), harvested at 48 hpi or 24 hpi respectively. Viral yields were determined by plaque assays. The histogram shows the mean values and standard deviations from biological replicates (N = 4). (C) HFFs transduced with lentivirus expressing SaCas9 and sgRNA were infected with HSV-1 at an MOI of 5 and harvested at 10 hpi. Proteins were detected using immunoblotting with antibodies specific for the indicated proteins. GAPDH was shown as a control. (D–G) HFFs transduced with lentivirus expressing SaCas9 and sgRNA were infected with HSV-1 at an MOI of 3 and harvested at the indicated time post infection. The accumulated DNAs were detected by real time PCR amplifying in the UL29 gene (ICP8, (D) or over the UL30-5 (E), UL26-27 (F), and UL37-38 (G) sgRNA target sites. The histogram shows the mean values and standard deviations from biological replicates (N = 4, (A and B) one-way ANOVA with Dunnett’s multiple comparisons test, (D–G) Ratio paired t test, *p<0.05, **p<0.01, and ***p<0.001).

Figure 8.

Figure 8—figure supplement 1. PCR amplification across the UL26-27 or UL37-38 target site in quiescent d109 genomes and reactivation.

Figure 8—figure supplement 1.

(A) Quiescently infected HFF cells were transduced with lentivirus expressing SaCas9/UL26-27 or SaCas9/UL37-38 sgRNA as described in Figure 2A and analyzed at the indicated sgRNA target sites by qPCR. qPCRs across the indicated sgRNA target sites were normalized to GAPDH. The histogram shows the mean values and standard deviations of biological replicates (N = 3). (B) Reactivation assay as described in Figure 3 using indicated sgRNAs (N = 6, Ratio paired t test, **p<0.01 and ***p<0.001).