Vectors based on adeno-associated viruses (AAV) have proven to be a convenient tool for genomic editing. However, for transgenesis of laboratory animals or gene therapy for human diseases, a high AAV titer is required. We optimized the conditions for producing AAV serotype 9 (AAV9) to increase the yield of the virus. A common approach is based on transfection of producer cells with three plasmids. We performed transfection with two plasmids encoding all the necessary components for AAV9 production. This allowed us to increase the final title fourfold. Optimization of the molar ratio of the plasmid containing the gene of interest to the plasmid encoding the proteins of the replicative complex, capsid, and auxiliary factors led to a twofold increase in the titer. Optimization of the composition of the cultivation medium for AAV9 production allowed us to increase significantly the yield of the virus. When the DMEM-F12 production medium with a less nutritious DMEM was supplemented with fetal bovine serum, the yield of AAV9 increased by approximately three orders of magnitude. Thus, optimization of the number and ratio of plasmids, as well as the composition of the cell culture medium, made it possible to increase significantly the production of AAV9 and achieve a final titer of 2.5 × 10¹² vector genomes in 1 mL of the production medium of the adhesive HEK293T cell line.

Background/Objectives: CRISPR/Cas-dependent base editing enables the precise correction of genomic sequences by direct modification of DNA bases without creating potentially mutagenic DNA double-strand breaks. Base editing is considered a therapeutic approach for rare monogenic disorders or viral infections. However, many variants of base editors suffer from limited specificity because they induce bystander edits within a broad editing window. However, nucleotide substitution is sufficient to correct a mutation or to achieve a therapeutic effect. Methods: PCR, cloning by restriction digest, and ligation were used to create genetic constructs. The editing efficiency was evaluated using NGS. Virus-like particles were produced in HEK293T cells using transient expression. VLPs were visualized by electron microscopy. Results: Using dual base editors that induce C-to-T and A-to-G substitutions, we show that high editing precision can be achieved by engineering the linker between the MmCas12m domain and the deaminase domain of the editor. By testing different linker sequences, we have created high-precision dual base editors with narrowed editing windows that can edit a single nucleotide at a target position with high accuracy. Conclusions: We found that shortening the linker to an acceptable length provides a single-nucleotide substitution in vitro with minimal bystander activity (less than 0.8%), while the high-precision MmCas12m dual base editor retains up to 90% of the editing efficiency of the original base editor. These high-precision base editors will be useful in cases where high precision is required, and editing of adjacent nucleotides is undesirable.

The use of base editors has been a significant achievement in the field of genomic editing. This approach involves the point-by-point editing of nitrogenou

Approaches to delivering gene editing tools in the form of ribonucleoproteins may provide a safety advantage over the delivery of nucleic acids encoding ribonucleoproteins. Virus-based vectors are widely used as a delivery platform. However, the persistence of viral exogenous nucleic acids can cause increased genotoxicity. Virus-like particles (VLPs) do not contain an expression cassette and can act as a platform for the delivery of ready-made ribonucleoprotein complexes. The absence of nucleic acids in VLPs eliminates the risk of insertional mutagenesis compared to widely used lentiviruses or adeno-associated viruses. Therefore, we used VLPs to deliver the ribonucleoprotein complex MmCas12m–TadDE to disrupt the HIV-1 gag gene start codon. We detected VLP morphogenesis using electron microscopy. We confirmed the incorporation of MmCas12m–TadDE into VLPs. We achieved an editing efficiency of about 9% in some cases with minimal off-target effects, which confirms the prospect of using VLPs as a platform for delivering genomic editing tools.