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

Figure 5. Effect of CRISPR-Cas9 on input and replicating HSV genomes.

(A) Kinetics of indel mutations in the HSV-1 genome during lytic infection. HFFs transduced with lentivirus expressing SaCas9 and sgRNA were infected with HSV-1 at an MOI of 3 in the presence or absence of phosphonoacetic acid (PAA) and harvested at the indicated times post infection. Indel mutation frequencies are shown at the indicated times post infection at the sgRNA target sites by MiSeq. (B) Calculated portion of short indels (≤±6 nucleotide (NT) indel) out of total indel during the lytic replication. (C and D) HFFs transduced with lentivirus expressing SaCas9 and sgRNA were infected with HSV-1 at an MOI of 3 and harvested at the indicated times post infection. The accumulated DNAs were detected by real time qPCR amplification within the UL29 gene (C) or over the UL30-5 sgRNA (D) target site. The histogram shows the mean values and standard deviations of biological replicates (N ≥ 3, Ratio paired t test, *p<0.05 and **p<0.01). Ligation-Mediated -PCR (LM-PCR) of HSV-1 viral DNA during lytic replication. (E) Schematic diagram of LM-PCR (adapted and modified from Brinkman et al., 2018). To make blunt end dsDNA, primer extension was performed using cleaved dsDNA and a primer (primer 1) about 500 bp away from the sgRNA target site. An annealed adaptor was ligated to the blunt end dsDNA and PCR amplification was performed using the pair of either the primer 1/adaptor primer or the primer 1/primer 2 for control. (F and G) HFFs transduced with lentivirus expressing SaCas9 and UL30-5 sgRNA were infected with HSV-1 (MOI of 3) in the absence (F) or presence (G) of PAA and harvested at the indicated times post infection. Total DNA was purified, and a primer extension reaction was performed using a complementing primer downstream of the UL30-5 site to convert all the cleaved DNA into blunt end dsDNA. An adaptor was ligated to the blunt end of dsDNA, and PCR was performed using a primer complementing adaptor and the primer used for the extension reaction. Top panel: +UL30-5 sgRNA, bottom: control. C: control PCR product generated using the primer for the extension reaction and a primer complementing near the UL30-5 target site. The right lane (M): DNA ladder.

Figure 5.

Figure 5—figure supplement 1. Histogram of indel length of HSV-1 and short indel accumulation during lytic replication.

Figure 5—figure supplement 1.

The lengths of SaCas9/UL30-5 sgRNA induced indel mutations in HSV-1 genome during lytic replication are analyzed using the Inference of CRISPR Editing (ICE) tool (https://ice.synthego.com) at indicated times post infection. (A–E) HFF cells were infected with HSV-1 and harvested at the indicated time post infection.