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. 2020 Sep 28;11(1):4884.
doi: 10.1038/s41467-020-18678-0.

Viral gene drive in herpesviruses

Marius Walter  1 , Eric Verdin  2
Affiliations

Viral gene drive in herpesviruses

Marius Walter et al. Nat Commun. .

Abstract

Gene drives are genetic modifications designed to propagate in a population with high efficiency. Current gene drive strategies rely on sexual reproduction and are thought to be restricted to sexual organisms. Here, we report on a gene drive system that allows the spread of an engineered trait in populations of DNA viruses and, in particular, herpesviruses. We describe the successful transmission of a gene drive sequence between distinct strains of human cytomegalovirus (human herpesvirus 5) and show that gene drive viruses can efficiently _target and replace wildtype populations in cell culture experiments. Moreover, by _targeting sequences necessary for viral replication, our results indicate that a viral gene drive can be used as a strategy to suppress a viral infection. Taken together, this work offers a proof of principle for the design of a gene drive in viruses.

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Conflict of interest statement

A patent application describing the use of a gene drive in DNA viruses has been filed by the Buck Institute for Research on Aging (Application number PCT/US2019/034205, pending, inventor: M.W.). E.V. declares no competing interests.

Figures

Fig. 1
Fig. 1. Gene drive in herpesviruses.
CRISPR-based gene drive sequences are, at a minimum, composed of Cas9 and a gRNA _targeting the complementary wildtype (WT) locus and can harbor an additional ‘cargo’ that will be carried over with the rest of the sequence. When present in the same cell nucleus, Cas9 _targets and cleaves the WT sequence. Homology-directed repair of the damaged WT locus using the gene drive sequence as a repair template ensures the conversion of the WT locus into a new gene drive sequence. In herpesviruses, gene drive involves the coinfection of a given cell by a WT and a modified virus. Cleavage and repair of the WT genome convert the virus into a new gene drive virus, spreading the modification into the viral population. In this example, hCMV WT virus (Towne strain) expresses eGFP florescent protein, and the gene drive virus (TB40/E strain) carries mCherry. Recombinant viruses then express both eGFP and mCherry.
Fig. 2
Fig. 2. Design of a gene drive system _targeting hCMV UL23.
a Modified and unmodified UL23 locus. Here, the gene drive cassette is composed of spCas9 followed by the SV40 polyA signal, an SV40 promoter driving an mCherry reporter, the beta-globin polyA signal and a U6-driven gRNA. b UL23 CRISPR cutting site and sequence of WT and modified viruses. spCas9 transcription is driven by UL23 viral promoter. CDS: coding sequence. c Localizations of mCherry and eGFP cassette on hCMV genomes. UL/US: unique long/short genome segments. d Replication of Towne-eGFP, TB40/E, and GD-mCherry viruses. Viral titers were measured by plaque assay over time. Titers are expressed in plaque forming unit (PFU) per mL of supernatant. Error bars represent standard error of the mean (SEM) between biological replicates. n = 5 (TB40/E), or n = 8 (Towne-eGFP and GD-mCherry). Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Recombination of the gene drive cassette into the wildtype genome.
Fibroblasts were coinfected with Towne-eGFP (WT) and GD-mCherry, and supernatant was used to infect fresh cells. a Representative examples of fluorescent viral plaques spreading on fibroblasts, expressing either eGFP only (left), mCherry only (middle), or both (right). Scale bar: 100 μm. b PCR for mCherry (upper band) and eGFP (lower band) on 48 recombinant viral genomes from two independent experiments. c Same as (b), after coinfection with GD-mCherry and a mutated Towne-eGFP (two biological replicates, 30 genomes). d PCR of homology arms and Sanger sequencing of 17 eGFP-mCherry expressing viral clones. Blue dots: single nucleotide polymorphisms (SNPs) from Towne strain; Red dots: TB40/E strain. e Fraction of SNPs of Towne or TB40/E origin alongside the hCMV genome. Each dot represents an individual SNP. Data combine two biological replicates after Oxford Nanopore sequencing. Coverage gives the number of reads, allowing multiple mapping. f Reconstruction of recombination history of individual hCMV genomes from long (>200 kb) Nanopore reads. One genome corresponds to one sequencing read and colors indicate the strain of origin. Gaps represent regions deleted compared to the reference genome, dashed lines indicate uncovered region. UL/US: unique long/short genome segments. GeneRuler 1 kb DNA ladder (ThermoFisher) was used in agarose gels as a size marker. The ladder is shown in the gel images, with the stronger band corresponding to 500 bp. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Gene drive sequences efficiently spread into the wildtype population.
a Coinfection experiments between Towne-eGFP (MOI = 0.1 at day 0) and different starting concentration of GD-mCherry: Viral titers (lower panels) and proportion (upper panels) over time of viruses expressing eGFP alone, mCherry alone, or both as measured by plaque assay. Left panel: 50% of GD-mCherry at Day 0, n = 6; middle: 10% at D0, n = 4; right: 0.1% at D0, n = 3. b Viral titers and proportion of viruses after coinfection with equal amount of Towne-eGFP (with a mutated _target site) and GD-mCherry. n = 6. c Viral titers and proportion of viruses after coinfection with equal amount of Towne-eGFP and GD-ΔCas9. n = 3. Titers are expressed in plaque forming unit (PFU) per mL of supernatant. Error bars represent standard error of the mean (SEM) between biological replicates. In the panels representing the viral population, data show both the mean and the individual trajectory of biological replicates. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Spread of a defective gene drive virus.
a Viral titers at day 10 in the presence of increasing concentrations of Interferon-γ (IFN-γ). n = 3 (IFN-γ = 100 ng/mL) or n = 5 (other concentrations). b Viral titer and proportion of viruses expressing eGFP alone, mCherry alone, or both, after coinfection with equal amounts of Towne-eGFP and GD-mCherry, in presence of increasing concentrations of IFN-γ. n = 3 (IFN-γ = 100 ng/mL) or n = 5 (other concentrations). c Supernatants from coinfected cells were collected at day 10 and used to infect fresh cells. Titers were measured 8 days later. Colors indicate the proportion of the different viruses relative to the height of the bar. n = 4. d Model for the spread of defective gene drive viruses: Wildtype viruses express UL23 and block IFN-γ antiviral response while gene drive viruses are UL23-KO and are severely inhibited by IFN-γ. Upon coinfection, UL23 originating from the wildtype virus—either brought in with the incoming virion as a tegument protein or expressed early on—is sufficient to block the IFN-γ antiviral response. A first generation of new gene drive viruses can be created. These viruses are, however, UL23-KO and are severely inhibited by IFN-γ when infecting new cells. Titers are expressed in PFU/mL. Error bars represent SEM between biological replicates. *p-value < 0.05; ****p < 0.0001; two-way ANOVA with Sidak’s multiple comparison test on log-transformed value. Source data are provided as a Source Data file.
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