Polyomaviruses are a family of small DNA tumor viruses that includes several pathogenic human members, including Merkel cell polyomavirus, BK virus and JC virus. As is characteristic of DNA tumor viruses, gene expression in polyomaviruses is temporally regulated into an early phase, consisting of the viral regulatory proteins, and a late phase, consisting of the viral structural proteins. Previously, the late transcripts expressed by the prototypic polyomavirus simian virus 40 (SV40) were reported to contain several adenosines bearing methyl groups at the N⁶ position (m⁶A), although the precise location of these m⁶A residues, and their phenotypic effects, have not been investigated. Here, we first demonstrate that overexpression of the key m⁶A reader protein YTHDF2 induces more rapid viral replication, and larger viral plaques, in SV40 infected BSC40 cells, while mutational inactivation of the endogenous YTHDF2 gene, or the m⁶A methyltransferase METTL3, has the opposite effect, thus suggesting a positive role for m⁶A in the regulation of SV40 gene expression. To directly test this hypothesis, we mapped sites of m⁶A addition on SV40 transcripts and identified two m⁶A sites on the viral early transcripts and eleven m⁶A sites on the late mRNAs. Using synonymous mutations, we inactivated the majority of the m⁶A sites on the SV40 late mRNAs and observed that the resultant viral mutant replicated more slowly than wild type SV40. Alternative splicing of SV40 late mRNAs was unaffected by the reduction in m⁶A residues and our data instead suggest that m⁶A enhances the translation of viral late transcripts. Together, these data argue that the addition of m⁶A residues to the late transcripts encoded by SV40 plays an important role in enhancing viral gene expression and, hence, replication. All organisms, including all viruses, use mRNAs to express the proteins required for their viability and replication. Viruses, in particular, are under pressure to replicate rapidly in order to spread before the host immune response becomes effective. Previously, it was reported that several viruses, including polyomaviruses, express mRNAs that bear methylated adenosine (m⁶A) residues, but the functional relevance of this modification was unclear. Here, we demonstrate that addition of m⁶A to mRNAs encoded by the prototypic polyomavirus SV40 increases both viral structural protein expression and viral spread. Targeting the sites of addition of m⁶A on SV40 mRNAs, using mutagenesis, therefore reduces SV40 replication, and a drug that globally inhibits m⁶A addition has a similar effect. These data identify m⁶A addition as a key step, and potential Achilles heel, in the polyomavirus life cycle.

CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann’s epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.

CRISPR/Cas9 holds immense potential to treat a range of genetic disorders. Allele-specific gene disruption induced by non-homologous end-joining (NHEJ) DNA repair offers a potential treatment option for autosomal dominant disease. Here, we successfully delivered a plasmid encoding S. pyogenes Cas9 and sgRNA to the corneal epithelium by intrastromal injection and acheived long-term knockdown of a corneal epithelial reporter gene, demonstrating gene disruption via NHEJ in vivo. In addition, we used TGFBI corneal dystrophies as a model of autosomal dominant disease to assess the use of CRISPR/Cas9 in two allele-specific systems, comparing cleavage using a SNP-derived PAM to a guide specific approach. In vitro, cleavage via a SNP-derived PAM was found to confer stringent allele-specific cleavage, while a guide-specific approach lacked the ability to distinguish between the wild-type and mutant alleles. The failings of the guide-specific approach highlights the necessity for meticulous guide design and assessment, as various degrees of allele-specificity are achieved depending on the guide sequence employed. A major concern for the use of CRISPR/Cas9 is its tendency to cleave DNA non-specifically at “off-target” sites. Confirmation that S. pyogenes Cas9 lacks the specificity to discriminate between alleles differing by a single base-pair regardless of the position in the guide is demonstrated.

The data presented in the present study demonstrate that MLV RNAs bear an exceptionally high level of the epitranscriptomic modifications m ⁶ A, m ⁵ C, and Nm, suggesting that these each facilitate some aspect of the viral replication cycle. Consistent with this hypothesis, we demonstrate that mutational removal of a subset of these m ⁶ A or m ⁵ C modifications from MLV transcripts inhibits MLV replication in cis , and a similar result was also observed upon manipulation of the level of expression of key cellular epitranscriptomic cofactors in trans . Together, these results argue that the addition of several different epitranscriptomic modifications to viral transcripts stimulates viral gene expression and suggest that MLV has therefore evolved to maximize the level of these modifications that are added to viral RNAs.