Un1Cas12f1 and Cas9 gene drive in HSV1: viruses that ‘infect’ viruses

Reviewer #1 (Public Review):

Summary:

The authors developed a new viral 'gene drive' based on an alternate CRISPR Cas system: UNCas12f1. They show in HSV-1 that the gene drive virus can transmit as hypothesized and is superior to Cas9 in terms of evolutionary robustness.

Strengths:

No doubt this is an impressive technological achievement and UNCas12f1 does appear superior to Cas9 in terms of taking longer to develop resistance. This is a strong body of work and Fig 3B is the crux of the paper for me showing that resistance does take longer in terms of % of viruses that are wildtype versus UNCas12f1 gene drive. I applaud the authors and I think this is a nice technological contribution.

Weaknesses:

I will focus on major conceptual issues.

(1) Mechanism. It is not really that clear to me why the UNCas12f1 has a higher barrier to the evolution of resistance. Is this simply a temporal delay or is there something intrinsic about UNCas12f1 that does not allow resistance to arise? There is a some discussion about this but it is speculative and I could not understand why resistance would not develop.

(2) Evolution. Fig 3B is the crux of the paper for me showing that resistance does take longer in terms of % of viruses that are wildtype versus UNCas12f1 gene drive. The authors did a nice job, however, I think they need to temper the claims somewhat as longer studies (other studies typically go out to >40 days) might show resistance arising. Also, I think absolute viral titers need to be shown in addition to percentage of viruses.

(3) Therapeutic Utility. Is this proposed as a therapeutic strategy? If so, how would it work? Could it lower overall total viral burden (i.e., wt + gene drive)? Another issue that I think needs to be specifically addressed is the issue of MOI as typically HSV-1 is thought to be (i.e. shown to be) a low MOI infection in vivo and in patients, whereas this strategy appears to rely on high MOI. Overall, to me, this is probably the major weakness: i.e., whether this strategy has therapeutic potential.

(4) Title. I don't think the subordinate clause of the title "virus that 'infect' viruses" is quite correct. This needs to be be reworded. This strategy converts the viral population from wild type to a gene drive virus but "infect" does not seem accurate.

Reviewer #2 (Public Review):

Summary:

This article develops CRISPR-based gene drives designed to spread in viral populations. By targeting the gene drives to neutral loci, or at least loci where the presence of a gene drive is tolerated. This type of gene drive is designed to work by recognising the cognate target sequence of the CRISPR-Cas nuclease on a wild type virus genome, cutting it and then invoking the homology-directed DNA repair machinery to copy itself into the repaired genome, thereby increasing its frequency in the population. Two types of CRISPR nuclease are tested in this setup: Cas9 and Cas12. There have been a large number of studies describing Cas9- based gene drives, but very few using other Cas nucleases, such as Cas12 reported here. Other nucleases have different targeting ranges and different features of cleavage that may make them more attractive for several reasons, including propensity to generate mutations that may be undesirable for certain applications. For this reason the work reported here is an important step.

There are advantages to this system, in terms of its throughput and speed of testing, which could generate insights into the dynamics of gene drive mutation and repair events. However, its suitability as a proxy for probability of selection of resistant mutations in gene drives designed to work in higher organisms is overstated since this is in large part determined by the force of selection acting on those mutations in the genomes of those target organisms.

Strengths:

Overall I found the experiments to be well planned and executed, with sound rationale and logic. The paper is well structured and well written. The evidence for CRISP-HDR in placing transgenes in specific parts of the viral genome is solid. The experiments to measure frequency of gene drive genotypes invading in the context of convertible WT target sites, and non-convertible target sites, are largely well designed. The authors go further and show in subsequent experiments that there are converted genotypes that contain combinations of linked alleles that should only segregate together in the event of conversion to the gene drive allele (assuming this signal is not conflated by two separate genotypes covering each other). The description of the different types and rates of accumulation of mutations according to Cas architecture is valuable.

Figures are very clear and informative (but could be improved with clearer labelling of genotypes).

The paper is well referenced and captures the literature well.

Weaknesses:

It is not immediately clear to me how you can determine, in your experimental setup, that the three alleles (gD+, GFP+ and gE-) are on the same genome/haplotype rather than split across two or more genomes that infect a cell. Presumably this is because you make a clonal population that started from a dilution that ensure there was at most one genome to start the infection?

Some more discussion of the results, and some surprising observations therein, is warranted. For example: in the invasion experiments, which are generally well described, it is curious that when nearly all the WT target sites are depleted there should still be a further disappearance of the original gene drive allele to the expense of the new converted drive alelle - once WT target sites are exhausted (e.g. V10 in Fig 3B), there are no more opportunities to convert, one would expect ration of green:yellow to stay the same (assuming equal fitness between genotypes)? In fact, the yellow genotype, having both gene drive and Us8 deletion, is expected to be less fit, is it not? So this result is surprising, yet not discussed.

It is not clear why general levels of mutation increase across the whole amplicon, regardless of proximity to target site? e.g by Passage 7 in the Cas12 lines , Fig3D and 3E). Not discussed. This may be due to the fact that their ratio to WT target sequences is inflated due to the presence of the non-mapped sequences but again, the origin of the not mapped sequences is itself not explained.

Gene drives could theoretically increase their frequency by 'destroying' or disabling other genotypes, for example if Cas-induced cleavage removed the cut genome, rather than converting it. Presumably this is what motivated the authors to try and get a concrete signal of converted genotypes rather than just increase in frequency of the original gene drive genotype. This possibility is never discussed.

Line 140 re: the use of refractory target sites to show that gene drive genomes do not increase in frequency when there is no opportunity for genomes to convert; I like this control but it should be noted that there is the possibility, albeit unlikely, that general UL-3/4 deletions compete better than WT generally, and that has not been tested here.

In some places, the description of genotypes rather than arbitrary, non-informative strain names would really help.

It is not obvious to me either where the 'unmapped reads' come from - it is stated that "gene drive viruses took over and interefrered with PCR, causing many unmapped NGS reads". I am not sure what is meant here, and besides, this doesn't explain why reads would be unmapped. If the gene drive allele were too large to be amplified then it should not contribute to sequences in the amplicon.

Re: HSV1 viruses being multiploid - for people, like me, whose virology is not very good, some more explanation would be useful - are you proposing that this happens on 'loose' viral genomes circulating within nucleus or cytoplasm of host cell, or within virions? Can there be more than one genome per virion?

The suggestion that slow reproduction in insects (where many types of gene drive are proposed for control of pest populations) is a barrier to testing at scale is only true to an extent - rue to an extent but there are screens for resistance that are higher throughput and do not need selection experiments over time, but rather in a single generation (e.g KaramiNejadRanjbar et al PNAS 2018; Hammond et al PLoS Genetics 2021) and, for the reasons stated above, selection on an insect genome cannot be replicated in this HSV system.

In the intro, much is made of utility in viral engineering for therapeutic approaches but there is never any detail of this in the discussion other than vague contemplations on utility in 'studying horizontal gene transfer' and 'prevention and treatment of diseases'.
I have other suggestions for improving clarity of text around experimental design but I have confined these to 'Recommendations for Authors'

Reviewer #3 (Public Review):

Summary:

The study by Yao, Dai and colleagues successfully describes the design of a viral gene drive against herpes simplex virus 1. Gene drives are genetic modifications designed to spread efficiently in a population. Most applications have been developed in insects to eradicate diseases such as malaria, and the design of gene drives in viruses is an exciting recent development. A viral gene drive system was first described with human cytomegalovirus, another virus of the herpesvirus family (PMID: 32985507), and the authors followed similar methods to design a gene drive against HSV-1. While some key experiments lack rigorous controls, overall the authors convincingly showed that an HSV-1 gene drive could spread efficiently in the target population in cell culture experiments. Cytomegalovirus and HSV-1 have very different infection dynamics, and these new findings suggest that viral gene drives could be developed in a wide variety of herpesviruses. This significantly expands the potential of the technology and will be of interest to readers interested in gene drives, viral engineering, or biotechnology in general.

The most novel and interesting part of the study is the comparison of gene drives relying on spCas9 and Un1Cas12f1 nuclease. Most gene drives developed to date have relied on Cas9 or similar nucleases. Cleavage and repair of the target site by non-homologous end-joining (NHEJ) can lead to the formation of drive-resistant sequences, and, depending on the selective pressure on the wild-type, gene drive and drive-resistant alleles, prevent successful gene drive propagation. By contrast to most RNA-guided nucleases, Un1Cas12f1 cleaves outside of the RNA-recognition site. The authors hypothesized that it could prevent the appearance of drive-resistant sequences, since the target sequence would be preserved after NHEJ repair. Indeed, the study convincingly showed that Un1Cas12f1 induced fewer drive-resistant mutations, which led to almost complete penetrance of the drive. However, the claim in the abstract that an "Un1Cas12f1 gene drive yielded a greater conversion" rate than Cas9 appears unsupported. Together with its smaller size, this positions Un1Cas12f1 as an interesting alternative to Cas9 for gene drives in any organism. This development will be of great interest to researchers interested in gene drives.

Strengths:

Overall, this study is well done and the main conclusions are supported by the data. The authors used flow cytometry to follow gene drive propagation, detecting either fluorescent or cell surface proteins expressed by the different viral populations. This represents an indirect but adequate way of measuring the proportion of the different viral populations, assuming that each of the target BHK cells is infected with only one virus.
In particular, the results in Fig 3 showing that Un1Cas12f1 induces fewer drive-resistant mutations than Cas9 are convincing.

Weaknesses:

The manuscript presents several conceptual and methodological weaknesses that could be discussed or addressed experimentally, which would improve the overall rigor of the study.

(1) In the abstract and the text, the author claims that "HSV1 emerges as a dependable and swift platform for gene drive assessment". It is unclear if the author believes that the main interest of their work with HSV-1 is to provide a platform for testing gene drive for other organisms, or whether a gene drive for HSV-1 could be useful by itself. While their findings with Un1Cas12f1 certainly warrant investigation in other systems, the dynamics of DNA cleavage, recombination, and selection of drive-resistant alleles will be very different between a viral infection where hundreds or thousands of genome copies co-exist in a cell nucleus, and during sexual reproduction where only one gene drive and wild-type allele are present in a fertilized egg. As such, it is unsure whether gene drive dynamics in HSV-1 will be informative for other organisms besides other herpesviruses. On the other hand, the authors provide little perspectives on the potential usage of an HSV-1 gene drive, beyond concluding that "Our study opens new possibilities for using the HSV1 gene drive for the prevention and treatment of diseases". The authors designed a drive against the important viral protein gE in an attempt to limit infectivity, but it is unclear from the data presented whether this was successful. An extended discussion on the potential use case of an HSV-1 gene drive would be informative.

(2) Unfortunately, the experiments presented lack rigorous controls to unambiguously show that gene drive propagation is mediated by CRISPR-directed recombination into the target genome. Gene drive-mediated recombination converts wild-type viruses into new recombinant viruses and the population of recombinants is expected to increase in frequency, as observed with the yellow population in Fig 2G and 3G. However, a rigorous experimental design would show that this population of recombinant viruses does not appear with a non-functional CRISPR system (for example if Cas9 is deleted in the gene drive virus) or if the target site is absent in the recipient virus. The comparison of Fig 2B and 2D does show that gene drive viruses do not increase in frequency when the target site is absent in the V19 virus, but these experiments could not distinguish between original and recombinant gene drive viruses. Thus, it is unknown if the increase in gene drive frequency in Fig 2B is because wild-type viruses have been converted to gene drive viruses, or because the WT and v23 viruses replicate with different dynamics (one could imagine for example that CRISPR cleavage of the WT genomes impaired the replication of the WT virus without inducing recombination, thus giving an advantage to v23). In Fig 2G and 3B, the authors do follow the population of recombinant viruses, in yellow, which increase in frequency as expected. However, in these experiments, either the donor or recipient viruses are mutated for gE, and the different viral populations might replicate with different dynamics, which confounds the interpretation of the results (see point 4. below). Overall, while the data presented suggests that CRISPR-mediated gene drive propagation is happening, it does not conclusively rule out other explanations, especially if viruses have different fitness.

(3) In Fig 2F-G-H, the authors designed a gene drive knocking out an important viral gene, gE, in an attempt to build a drive that reduces infectivity. gE knockout viruses V10 and V15 had smaller plaques but replicated with similar titers (Fig 1B, 1C). The gene drive against gE spread efficiently in Fig 2G. However, gE-KO viruses did not appear to have a meaningful disadvantage in the experimental system used, since the high MOI used in the co-infection experiments allowed to bypass the cell-to-cell defect of gE mutants. It would have been interesting to characterize the final population composed primarily of original and recombinant viruses (at P3 in Fig 2G), and in particular measure the plaque size of these viruses. Recombinant viruses should have smaller plaque sizes, and showing that the gene drive was able to propagate an attenuating phenotype would be a meaningful result that hints at potential therapeutic applications.

(4) Experiments presented in Fig 3 compared the dynamics of Cas9 and Un1Cas12f1 gene drives, but the experimental system used is a bit puzzling and makes the interpretation of the results challenging. In particular, the authors chose to use gE-knockout virus v10 as the recipient for the gene drive, which allowed them to use gE in their flow cytometry assay. Unfortunately, this added a confounding factor to the experiments, since gE- viruses might replicate with different dynamics than gE+ viruses (for example v10 titers are one log higher than WT at 12h in Fig 1C). In Fig 3B, gD+ gE- viruses (in blue) disappear and are replaced by gD+ GFP+ gE- recombinants (in yellow), which is suggestive of efficient gene drive recombination, as pointed out by the authors. However, the population of gD+ GFP+ virus (in green) representing the original gene drive virus also disappeared over time. At the end of the experiments in Fig 3B, the population of gE+ viruses is gone. This is unexpected and suggests that the gD+ GFP+ gE- (yellow) has a replicative advantage over gD+ GFP+ (green), and that the gE- mutation is actually positively selected in these viral competition assays. So in these experiments, both gene drive-mediated recombination and competition between viral genotypes appear to be happening at the same time, which makes interpretation of the results challenging. However, despite these limitations, the results presented convincingly suggested that Un1Cas12f1 gene drives achieved higher penetrance than Cas9's, which is one of the most important findings of the study.