PKD2基因敲除HEK293细胞

Mutations in the co-chaperone DNAJB11 have been shown to cause polycystic kidney disease. The molecular mechanism underlying DNAJB11-related kidney disease involves impaired processing of Polycystin-1 (PC1), the protein most commonly mutated in autosomal dominant polycystic kidney disease (ADPKD). Chaperones are known to form multiprotein complexes to facilitate folding and processing of client proteins. Yet, it is unknown whether DNAJB11 forms complexes with other proteins that are required for PC1 processing. In this study, we perform an unbiased interaction proteomics screen for DNAJB11-interacting proteins. We identify two highly homologous proteins, SDF2 and SDF2L1, as strong interaction partners of DNAJB11. Using newly established knockout cell lines, we demonstrate a reciprocal interdependence of DNAJB11 and SDF2/SDF2L1 protein abundance. Furthermore, we show that concomitant loss of SDF2 and SDF2L1 impairs PC1 processing, mimicking the biochemical phenotype caused by loss of DNAJB11. Using a combination of knockout cell lines and reexpression of the respective members of the DNAJB11 protein complex, we show that SDF2 or SDF2L1 are elementary subunits of the DNAJB11 complex required for normal PC1 processing.

Recent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.