Rapid target validation in a Cas9-inducible hiPSC derived kidney model

Abstract

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.

Figure 1

Differentiation of hiPSC into kidney organoids. (A) The schematic outline for the multi-stage kidney organoid differentiation is shown. (B) The resulting kidney organoids (day31) stained and imaged using confocal microscopy for DAPI and kidney specific markers including WT1, LTL and ECAD for podocyte, proximal tubule and distal tubule, respectively.

Figure 2

Validation of sgRNAs against PKD1 and PKD2. (A) Schematic presentation of the target sites from previous report and our study is shown. (B) Outline of the experimental procedure used for validation of the sgRNAs. Cells were seeded in 96-well format and Cas9 expression was induced before transfection with sgRNA-expressing plasmids. (C) The resulting Cas9-induced mutations at the target sites were measured using deep targeted sequencing. Mutations were further analyzed for their effect on the reading frame.

Figure 3

Paired-sgRNA strategy resulted in higher indel rate in hiPSCs. (A) Schematic outline of the experiment is shown. (B) The percentage of the modified reads for on-target loci were measured using deep targeted sequencing. (C) No Cas9 off-target activity was detected at the selected off-target sites. Samples were analyzed using deep targeted sequencing.

Figure 4

Cystic phenotype in kidney organoids using genetic perturbation. (A) Schematic outline of differentiation stages for conventional clonal knockout isolation and differentiation and speedy/direct differentiation of KO pools. (B) Cystic phenotype observed in organoids obtained from genetic perturbation of PKD1 and PKD2. (C) The resulting kidney organoids were stained and imaged using confocal microscopy for different markers. (D) Quantification of cyst formation rates in PKD KO pool organoids versus isogenic unmodified organoids (CTRL) as an average of all experiments. Data are shown as Mean. Bar represent standard deviation (SD) (n = 5 separate experiments).