1. Genetics and Genomics
  2. Microbiology and Infectious Disease
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Herpesviral lytic gene functions render the viral genome susceptible to novel editing by CRISPR/Cas9

  1. Hyung Suk Oh
  2. Werner M Neuhausser  Is a corresponding author
  3. Pierce Eggan
  4. Magdalena Angelova
  5. Rory Kirchner
  6. Kevin C Eggan
  7. David M Knipe  Is a corresponding author
  1. Blavatnik Institute, Harvard Medical School, United States
  2. Harvard University, United States
  3. Beth Israel Deaconess Medical Center, Harvard Medical School, United States
  4. Harvard TH Chan School of Public Health, United States
  5. Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, United States
Research Article
Cite this article as: eLife 2019;8:e51662 doi: 10.7554/eLife.51662
10 figures, 2 tables, 2 data sets and 1 additional file

Figures

In vitro cleavage assay.

(A) Schematic diagram of in vitro cleavage assay (B) Results are shown for three sgRNAs targeting UL30 (UL30-3, -4, and -5). T7 in vitro transcribed sgRNA was combined with SpCas9 protein and a PCR template containing the CRISPR sequence, incubated 1 hr at 37°C and run on an agarose gel. Lane (1) SpCas9+sgRNA, lane (2) Cas9 only, lane (3) sgRNA only, lane (4) no Cas9/sgRNA. Efficient cutting is seen for UL30-4 and -5 but not UL30-3.

Effect of CRISPR-Cas9 on HSV-1 lytic infection.

(A) Experimental scheme of SaCas9/sgRNA-mediated inhibition of HSV lytic infection. (B and C) HFFs transduced with lentivirus expressing SaCas9 and sgRNAs were infected with HSV-1 at an MOI of 0.1 (C) or 5 (D) and harvested at 48 hpi or 24 hpi, respectively. Viral yields were determined by plaque assays. The histogram shows the mean values and standard deviations of biological replicates at an MOI of 0.1 (N = 3) or at an MOI of 5 (N = 4). All the sgRNA added conditions showed statistical significance compared to +Cas9 /-gRNA (one-way ANOVA with Dunnett’s multiple comparisons test, p<0.0001 (MOI of 0.1) and p<0.01 (MOI of 5, except for UL29-4 (p<0.05))). (D) 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. Three SDS-PAGE gels loaded with the same amount of proteins were used to detect multiple proteins. Immunoblots of GAPDH are shown as a control under the individual immunoblots.

CRISPR/Cas9-induced mutagenesis of quiescent d109 genomes and effect on reactivation.

(A) Experimental scheme of SaCas9/sgRNA-mediated inhibition of reactivation of quiescent d109 genomes in HFFs. HFFs were infected with HSV-1 d109 virus to establish quiescent infection for 7–10 d and transduced with lentivirus expressing SaCas9 and sgRNAs for 7–10 d. (B and C) HFF were infected with d109 to establish quiescent infection for 7–10 d and transduced with lentivirus expressing SaCas9 and sgRNAs for 7–10 d. To reactivate quiescent d109 genomes, HFFs were superinfected with WT HSV-1 at an MOI of 5 and harvested at 24 hpi. GFP-positive viral yields were determined by plaque assays on FO6 and V27 cells. The histogram shows the mean values and standard deviations of biological replicates (B and C: N = 5 and N = 7 respectively). All the sgRNA added conditions showed statistical significance compared to +Cas9 /-gRNA (Ratio paired t test, *p<0.05 and **p<0.01).

Indel mutations in the HSV-1 genome during the quiescent infection.

(A) Indel mutation frequencies of quiescent d109 genomes are shown at the indicated sgRNA target sites. (B) Histogram representing the frequency (count) of indel lengths induced by SaCas9/UL30-5 in quiescent d109 genomes. (<0) deletions (>0) insertions. (C) Examples of sequences that show mutations induced by SaCas9/UL30-5 in quiescent d109 genomes.

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

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.

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).

On-target activity of UL30-5 sgRNA within the HSV genome sequence during lytic replication.

Per-base plot of WGS coverage over each specific base in HSV during lytic replication in HFFs for UL30-5 against untreated controls (no sgRNA, no SaCas9/sgRNA). (A) Per-base WGS coverage across the entire HSV genome with fitted LOESS curves obtained by local regression of per-base data (B) Zoomed in view of the WGS coverage in the genomic area around the UL30-5 cleavage site within UL30 for UL30-5 treated against untreated controls. The vertical lines demarcate the UL30-5 target sequence in each sample.

Figure 7—source data 1

List of 437 possible UL30-5 off-target sites within the human genome (GRCh38/hg38).

The 437 possible UL30-5 off-target sites are shown together with the off-target sequence and the number of mismatches at each position. Off-target sites were identified through sequence analysis using Cas-OFFinder to identify all predicted off-target sites for SaCas9/UL30-5 with ≤6 mismatches within GRCh38/hg38.

https://cdn.elifesciences.org/articles/51662/elife-51662-fig7-data1-v2.xlsx
Figure 8 with 1 supplement
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 supplement 1
PCR amplification across the UL26-27 or UL37-38 target site in quiescent d109 genomes and reactivation.

(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).

Effect of ICP0 on CRISPR-Cas9-induced DNA repair of input HSV genome.

HFFs transduced with lentivirus expressing SaCas9 and sgRNA were infected with ICP0-null mutant HSV-1 at an MOI of 3 in the presence of PAA and harvested at the indicated time post infection. The accumulated DNAs were detected by real-time qPCR amplifying within the UL29 gene (A) or over the UL30-5 sgRNA (B) target site. The histogram shows the mean values and standard deviations biological replicates (N ≥ 3, Ratio paired t test, *p<0.05 and **p<0.01).

Model for CRISPR/Cas9 mediated inhibition of HSV lytic replication, editing of latent HSV genomes, and inhibition of reactivation of latent HSV.

Lytic infection: Cas9/sgRNA cleaves input viral DNA. In the absence of viral DNA replication, either prior to the onset of viral replication or in the presence of PAA, the expression of Cas9/sgRNA targeting viral gene encoded protein is reduced and the input viral DNAs decrease. Cas9/sgRNA induces low levels of indel mutations at the sgRNA target site of the input viral DNA, and cleaved input viral DNA is accumulated. During viral DNA replication, expression of Cas9/sgRNA targeting the viral gene encoded protein is reduced, non-mutated template and its replicated viral DNAs are targeted by SaCas9/sgRNA, which results in a decrease of viral DNA, an increase in indel mutations and accumulation of cleaved viral DNA. Viral protein ICP0 contributes to Cas9/sgRNA-mediated editing/cleavage by removing histones and preventing DNA repair. Quiescent infection: Cas9/sgRNA induces indel mutations to viral DNA without significant change in latent viral DNA levels. (⬤: nucleosome) Reactivation: Cas9/sgRNA induces more indel mutations in non-mutated viral DNA and accumulation of cleaved viral DNA, which results in decrease of the expression of SaCas9/sgRNA targeted gene encoded protein, viral DNA, and viral replication. (⇧: increase, ⬇: decrease, ●: no change).

Tables

Table 1
CRISPR/Cas9 target sequences.
NameEfficiency of cleavageSaCas9 sgRNA + PAM
(g was added as needed)
Target sequences
(GenBank: KT899744)
UL30
UL30-1+GCGTCCCGACTGGGGCGAGGT AGGGGT62811–62831
UL30-2++gAAGTTTTGCCTCAAACAAGGC GGGGGT62779–62799
UL30-3-GCGGCGTGGACCACGCCCCGG CGGGGT63060–63080
UL30-4++gTGCCCCCCCGGAGAAGCGCG CCGGGGT62923–62942
UL30-5++gACACGTGAAAGACGGTGACG GTGGGGT63097–63116
UL30-6+gACCAGCCGAAGGTGACGAAC CCGGGGT63595–63614
UL30-7++GGCCATCAAGAAGTACGAGGG TGGGGT63532–63552
UL30-24*++gAAACCCCAAAAGCCGCTTGGG TGGGAT62589–62609
UL30-25*+gCCACCCGAACCCCTAAAGAGG GGGGAT62637–62657
UL30-26*-gCATGCCGGCCCGGGCGAGCCT GGGGGT62542–62562
UL30-27*++gCCATCCCACCCAAGCGGCTTT TGGGGT62581–62601
UL29
UL29-1++gTCAAGGTCCCCCCCGGGCCCC TGGGAT61861–61881
UL29-2+GTGTTTGAGGTCGCCGGGCCG GGGGGT61502–61522
UL29-3++GCCAGCCAGGGTAAGACCCCG CGGGGT61028–61048
UL29-4++GCCGCCGTCGCGCCCACCCCG CGGGGT61007–61027
UL29-14*++GAGGGTGGGAGACCGGGGTTG GGGAAT62029–62049
UL29-15*++GTCGGGCGTCCGTCGTCGTGC CGGGAT61952–61972
UL29-16*++gCGGGGGTTGTCTGTGAAGGGT AGGGAT62064–62084
UL29-17*++gATCGGCACCCCGTGGTTACCC GGGGGT62084–62104
UL29-18*++gCAGACAACCCCCGGGTAACCA CGGGGT62072–62092
UL29-19*++GGACCCCGCGTTGCCAGCCGC CGGGGT62113–62133
UL29-20*++GAACCCCGGCGGCTGGCAACG CGGGGT62105–62125
UL29- 21++GGTTCTCGCACGACGGGGCTC GGGGGT61685–61705
UL54
UL54-1*++GCTGTCGGCTGCCGTCGGGGC TGGGGT113541–113561
UL54-2++gACCTGGAATCGGACAGCAAC GGGGAGT113667–113687
UL54-3++GCTCCGGTCCGTCCTCTCCGT GGGGGT113728–113748
UL54-4++GCGTCTGGGTGCTGGGTACGC CGGGGT113803–113823
UL54-5++GGCGGACGCCGTGGGCGTCGC AGGGGT113982–114002
UL54-6++gTGGTTCTGGGGGCACGCCGGC GGGGGT114055–114075
UL54-7++GCAGGCTGGGCTTTGGTCGGT GGGGGT113957–113977
UL54-8++gCGCCGTGGGCGTCGCAGGGGT CGGGGT113988–114008
UL54-9+GTCCGTCCACCCCGCCCCGGGG CGGGGT114098–114119
UL54-14*++gCGCTTCCGCGGGGACCCGGGC GGGGGT113234–113254
UL54-15*++gCGCCCGGGGGGCGGAACTAGG AGGGGT113347–113367
UL54-16++GGCGGCTCTCCGCCGGCTCGG GGGGGT113641–113661
Rs1
Rs1-1++GCCGGGCGTCGTCGAGGTCGT GGGGGT130775–130795, 146992–147012
Rs1-2-gCCGCTCGTCGCGGTCTGGGCT CGGGGT130866–130886, 146901–146921
Rs1-3-GGGGGTGGTCGGGGTCGTGGT CGGGGT130796–130816, 146971–146991
Rs1-4++gATCGTCGTCGGCTAGAAAGGC GGGGGT130599–130619, 147168–147188
Rs1-5++GGCGCGGCGACAGGCGGTCCG TGGGGT130475–130495, 147292–147312
Rs1-6++GCGAGGCCGCGGGGTCGGGCGT CGGGAT130634–130655, 147132–147153
Rs1-7+GGGTCCGGGGCGGCGAGGCCG CGGGGT130622–130642, 147145–147165
Rs1-8++gCGCGAGGCGCGGGCCGTCGGG CGGGGT130290–130310, 147477–147497
Rs1-9++GCGGACGACGAGGACGAGGACC CGGAGT130378–130399, 147388–147409
Rs1-15*++GCCGATGCGGGGCGATCCTCC GGGGAT130954–130974, 146813–146833
Rs1-16*-gTACGCGGACGAAGCGCGGGAG GGGGAT131142–131162, 146625–146645
Rs1-17*+gCGCGTCGACGGCGGGGGTCGT CGGGGT131061–131081, 146706–146726
Rs1-18*++GCGCTAGTTCCGCGTCGACGGC GGGGGT131070–131091, 146696–146717
UL26-27++GAGGAAATCGGCACTGACCAA GGGGGT52742–52762
UL37-38++GTATAACACCCCGCGAAGACG CGGGGT84066–84086
  1. ++:full cleavage (no residual substrate DNA on agarose gel), +: partial cleavage (some residual substrate DNA on agarose gel), -: no cleavage, *: non-coding region

Key resources table
Reagent type (species)
or resource
DesignationSource or
reference
IdentifiersAdditional information
Gene (Staphylococcus aureus)SaCas9AddgenepX601,
Cat. #: #61591
Peptide, recombinant proteinSpCas9NEBCat. #: M0386
Cell line (Homo-sapiens)HFF (Hs27)ATCCCat# CRL-1634,
RRID:CVCL_0335)
Cell line (Homo-sapiens)U2OSATCCCat# HTB-96,
RRID:CVCL_0042
Cell line (Chlorocebus sabaeus)VeroATCCCat# CCL-81,
RRID:CVCL_0059
AntibodyAnti-ICP8
(Rabbit serum)
Knipe et al., 19871:5000
AntibodyAnti-ICP4
(Mouse monoclonal, purified from
hybridoma cell
line 58S
(ATCC HB8183))
Showalter et al., 19811:2000
AntibodyAnti-ICP27
(Mouse monoclonal)
Eastcoast BioCat. #: P11191:5000
AntibodyAnti-GAPDH
([6C5],
Mouse monoclonal)
AbcamCat. #: ab82451:10000
Recombinant DNA reagentAddgenelentiCRISPRv2, Cat #: 52961Cloning vector
Recombinant DNA reagentpX601-AAV-CMV-SaCas9-T2A-mCherryThis paperTemplate of SaCas9-T2A-mCherry for
lentiSaCas9-mCherry-Puro
Recombinant
DNA reagent
lentiSaCas9-mCherry-PuroThis paperCloned SaCas9 gene into lentiCRISPRv2
Software, algorithmICE analysis
toolbox
https://ice.synthego.com
Software, algorithmbcbio-nextgenhttps://github.com/bcbio/bcbio-nextgenv1.15
Software, algorithmMuTect2https://www.ncbi.nlm.nih.gov/pubmed?term=20644199v2
Software,
algorithm
bwa-memhttps://arxiv.org/abs/1303.3997v0.7.17
Software, algorithmCas-OFFinderhttps://www.ncbi.nlm.nih.gov/pubmed/24463181v2.4

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

The following previously published data sets were used
  1. 1
    NCBI GenBank
    1. RC Colgrove
    2. X Liu
    3. A Griffiths
    4. P Raja
    5. NA Deluca
    6. RM Newman
    7. DM Coen
    8. DM Knipe
    (2016)
    ID KT899744.1. Human herpesvirus 1 isolate KOS, complete genome.
  2. 2
    Genome Reference Consortium
    1. KH Miga
    2. Y Newton
    3. M Jain
    4. N Altemose
    5. HF Willard
    6. WJ Kent
    (2014)
    ID Human GRCh38.p12 (GCA_000001405.27). hg38.

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