Efficient and Programmable Particle Delivery for Genome Editing in Human Kidney Organoids

Background: Kidney organoids are complex structures that resemble nephrons and can be used to develop gene therapy approaches. Gene editing in organoids, much like in tissues, has great potential but currently suffers from inefficiency and lack of specificity. There is a critical need for the development of methods for efficient and precise genome editing. Methods: We developed CaSh (Calcium Shock), a method that improves transfection and transduction, and CaSh-Pro, a programmable version that targets specific cell types. To enable better detection of genome editing events, we developed a fluorescence-on (tdTomato) reporter system in human iPS cells. Intact kidney organoids were transfected with CRISPR ribonucleoprotein (RNP) complexes. To improve delivery, identified molecular targeting agents (MTAs) specifically recognizing podocytes, proximal tubules, or endothelial cells in live imaging assays, and tethered these to Cas9. Genome editing events or adeno-associated virus (AAV) transduction were detected by changes in fluorescence in specific cell types. Results: CaSh dramatically improved genome editing by CRISPR-Cas9 ribonucleoprotein from ~ 2% in organoids without CaSh to 15% with CaSh. CaSh-Pro, which incorporates specific MTAs, resulted in substantially enhanced editing in the targeted cell types of podocytes (~12%) (Figure 1) or tubules (~5%), as detected by tdTomato co-localization with a specific marker, relative to negative controls including untreated, no MTA, or generic IgG. Finally, CaSh improved transduction of AAV8 encoding a red fluorescent reporter protein into proximal tubular epithelial cells, resulting in ~15% efficiency at 1 x 105 genome copies (gc)/cell, compared to negative controls. Conclusion: CaSh and CaSh-Pro provide versatile protocols for biological discovery, therapeutics development, and genome editing. Our innovative method improves specificity and efficiency enabling a wide variety of potential applications such as testing gene therapies in specific cell types as a surrogate for tissues. Gene editing can be achieved in a clinically effective range (> 10%) and in specific cell types, enabling future studies of phenotypic rescue in human models. Funding: NIDDK Support