Capsid-mediated control of adeno-associated viral transcription determines host range.

Adeno-associated virus (AAV) is a member of the genus Dependoparvovirus, which infects a wide range of vertebrate species. Here, we observe that, unlike most primate AAV isolates, avian AAV is transcriptionally silenced in human cells. By swapping the VP1 N terminus from primate AAVs (e.g., AAV8) onto non-mammalian isolates (e.g., avian AAV), we identify a minimal component of the AAV capsid that controls viral transcription and unlocks robust transduction in both human cells and mouse tissue. This effect is accompanied by increased AAV genome chromatin accessibility and altered histone methylation. Proximity ligation analysis reveals that host factors are selectively recruited by the VP1 N terminus of AAV8 but not avian AAV. Notably, these include AAV essential factors implicated in the nuclear factor κB pathway, chromatin condensation, and histone methylation. We postulate that the AAV capsid has evolved mechanisms to recruit host factors to its genome, allowing transcriptional activation in a species-specific manner.

Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing.

Type VI CRISPR enzymes have been developed as programmable RNA-guided Cas proteins for eukaryotic RNA editing. Notably, Cas13 has been utilized for site-targeted single base edits, demethylation, RNA cleavage or knockdown and alternative splicing. However, the ability to edit large stretches of mRNA transcripts remains a significant challenge. Here, we demonstrate that CRISPR-Cas13 systems can be repurposed to assist trans-splicing of exogenous RNA fragments into an endogenous pre-mRNA transcript, a method termed CRISPR Assisted mRNA Fragment Trans-splicing (CRAFT). Using split reporter-based assays, we evaluate orthogonal Cas13 systems, optimize guide RNA length and screen for optimal trans-splicing site(s) across a range of intronic targets. We achieve markedly improved editing of large 5' and 3' segments in different endogenous mRNAs across various mammalian cell types compared to other spliceosome-mediated trans-splicing methods. CRAFT can serve as a versatile platform for attachment of protein tags, studying the impact of multiple mutations/single nucleotide polymorphisms, modification of untranslated regions (UTRs) or replacing large segments of mRNA transcripts.

Epigenetic Silencing of Recombinant Adeno-associated Virus Genomes by NP220 and the HUSH Complex.

The single-stranded DNA genome of adeno-associated viruses (AAV) undergoes second-strand synthesis and transcription in the host cell nucleus. While wild-type AAV genomes are naturally silenced upon integration into the host genome, recombinant AAV (rAAV) genomes typically provide robust expression of transgenes persisting as extrachromosomal DNA or episomes. Episomal DNA associating with host histones is subject to epigenetic modifications, although the mechanisms underlying such are not well understood. Here, we provide evidence that the double-stranded DNA binding protein NP220, in association with the human silencing hub (HUSH) complex, mediates transcriptional silencing of single-stranded as well as self-complementary rAAV genomes. In cells lacking NP220 or other components of the HUSH complex, AAV genome transcript levels are increased and correlate with a marked reduction in repressive H3K9 histone methylation marks. We also provide evidence that the AAV capsid (serotype) can profoundly influence NP220-mediated silencing of packaged genomes, indicating potential role(s) for capsid-genome or capsid-host factor interactions in regulating epigenetic silencing of rAAV genomes. IMPORTANCE Recombinant AAV vectors can enable long-term gene expression in a wide variety of tissues. However, transgene silencing has been reported in some human gene therapy clinical trials. Here, we demonstrate the HUSH complex can suppress transcript formation from rAAV vector genomes by epigenetic modification of associated host histones. Further, the AAV capsid appears to play an important role in this pathway. We postulate that modulation of epigenetic pathways could help improve rAAV expression.