Rationally Engineered Miniaturized Plasmids Improve Transgene Delivery and CRISPR Activation of PKD1 in Primary Kidney Cells

Background: Loss-of-function mutations in PKD1 are the major cause of autosomal dominant polycystic kidney disease (ADPKD). We hypothesized that increasing the transcription of PKD1 using CRISPR activation (CRISPRa) would increase polycystin-1 levels in primary cyst epithelial cells and rescue a normal cellular phenotype. However, efficient delivery of large genetic constructs into primary kidney cells is technically challenging, with transfection efficiency declining sharply with an increase in vector size. While viral vectors are often considered, their limited cargo capacity, need for high titers, and poor infectivity in renal epithelial cells restrict their utility. To overcome these challenges, we developed a synthetic strategy to miniaturize expression constructs, thereby improving delivery and expression efficiency in primary kidney cells. Methods: We designed plasmids containing Type IIS restriction sites with complementary overhangs flanking the gene of interest (GOI). Iterative cycles of digestion and ligation led to selective recircularization of the GOI, while linear DNA was degraded using T5 exonuclease. These “mini plasmids” were first tested using fluorescent reporters to assess uptake efficiency. We then miniaturized large CRISPRa constructs targeting the endogenous PKD1 locus and evaluated their delivery and activity in primary mouse and human kidney epithelial cells, including both normal and cystic cells. Results: Mini plasmid synthesis achieved a 40.2%–66.5% reduction in vector size. When electroporated into primary kidney cells, mini plasmids demonstrated a 1.5- to 2.5-fold increase in transfection efficiency (corresponding to a 40%–150% increase in uptake) compared to full-length plasmids. Miniaturized CRISPRa constructs achieved ~2- to 3.5-fold upregulation of endogenous PKD1 expression, levels that were not attainable with the original constructs. Conclusion: Our vector miniaturization approach significantly enhances the delivery of large constructs into hard-to-transfect primary kidney cells. Our method offers an efficient and scalable strategy to enable gene modulation studies such as CRISPRa in primary kidney cells, overcoming the insufficiency of traditional large plasmid delivery. Funding: Private Foundation Support