rAAV capsid mutants eliminate leaky expression from DNA donor template for homologous recombination.

Precise genomic editing through the combination of CRISPR/Cas systems and recombinant adeno-associated virus (rAAV)-delivered homology directed repair (HDR) donor templates represents a powerful approach. However, the challenge of effectively suppressing leaky transcription from the rAAV vector, a phenomenon associated to cytotoxicity, persists. In this study, we demonstrated substantial promoter activities of various homology arms and inverted terminal repeats (ITR). To address this issue, we identified a novel rAAV variant, Y704T, which not only yields high-vector quantities but also effectively suppresses in cis mRNA transcription driven by a robust promoter. The Y704T variant maintains normal functionality in receptor interaction, intracellular trafficking, nuclear entry, uncoating, and second-strand synthesis, while specifically exhibiting defects in transcription. Importantly, this inhibitory effect is found to be independent of ITR, promoter types, and RNA polymerases. Mechanistic studies unveiled the involvement of Valosin Containing Protein (VCP/p97) in capsid-mediated transcription repression. Remarkably, the Y704T variant delivers HDR donor templates without compromising DNA replication ability and homologous recombination efficiency. In summary, our findings enhance the understanding of capsid-regulated transcription and introduce novel avenues for the application of the rAAV-CRISPR/Cas9 system in human gene therapy.

Role of Flaxseed Gum and Whey Protein Microparticles in Formulating Low-Fat Model Mayonnaises.

Flaxseed gum (FG) and whey protein microparticles (WPMs) were used to substitute fats in model mayonnaises. WPMs were prepared by grinding the heat-set whey protein gel containing 10 mM CaCl2 into small particles (10-20 µm). Then, 3 × 4 low-fat model mayonnaises were prepared by varying FG (0.3, 0.6, 0.9 wt%) and WPM (0, 8, 16, 24 wt%) concentrations. The effect of the addition of FG and WPMs on rheology, instrumental texture and sensory texture and their correlations were investigated. The results showed that all samples exhibited shear thinning behavior and 'weak gel' properties. Although both FG and WPMs enhanced rheological (e.g., viscosity and storage modulus) and textural properties (e.g., hardness, consistency, adhesiveness, cohesiveness) and kinetic stability, this enhancement was dominated by FG. FG and WPMs affected bulk properties through different mechanisms, (i.e., active filler and entangled polysaccharide networks). Panellists evaluated sensory texture in three stages: extra-oral, intra-oral and after-feel. Likewise, FG dominated sensory texture of model mayonnaises. With increasing FG concentration, sensory scores for creaminess and mouth-coating increased, whereas those of firmness, fluidity and spreadability decreased. Creaminess had a linear negative correlation with firmness, fluidity and spreadability (R2 > 0.985), while it had a linear positive correlation with mouth-coating (R2 > 0.97). A linear positive correlation (R2 > 0.975) was established between creaminess and viscosity at different shear rates/instrumental texture parameters. This study highlights the synergistic role of FG and WPMs in developing low-fat mayonnaises.