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Abstract: PO1325

An Off-the-Shelf CRISPR Gene Therapy Approach in Human Kidney Organoids

Session Information

Category: Genetic Diseases of the Kidneys

  • 1002 Genetic Diseases of the Kidneys: Non-Cystic


  • Vo, Nicole, University of Washington, Seattle, Washington, United States
  • Macnary, Catherine Agnes, University of Washington, Seattle, Washington, United States
  • Freedman, Benjamin S., University of Washington, Seattle, Washington, United States

Group or Team Name

  • The Freedman lab

Gene therapy offers many opportunities to treat kidney diseases. Targeted, off-the-shelf therapeutics are needed for both loss-of-function (e.g. nephropathic cystinosis) and gain-of-function (e.g. APOL1) disease states. Kidney organoids are complex structures that resemble nephrons and can be used to develop gene therapy approaches. Commonly used gene transfer techniques, such as lentivirus and adeno-associated virus, are size limited, transient, or introduce DNA non-specifically into the genome. While targeted CRISPR gene editing is routinely used in 2D cell cultures, it has been challenging to use this powerful technique in intact organoids.


To achieve off-the-shelf gene transfer, organoids were transfected with Cas9 and gRNA ribonucleoprotein (RNP) complexes targeting the AAVS1 safe harbor locus supplemented with knock-in cassettes encoding green fluorescent protein (GFP) or FLAG-tagged cystinosin (deficient in nephropathic cystinosis). Alternatively, to monitor gene knock-out, organoids expressing GFP from AAVS1 were transfected with RNP and either one or two gRNAs to introduce indels in the coding sequence. Genome editing was detected three ways: by confocal microscopy, PCR, and next generation sequencing.


GFP and cystinosin knock-in events in organoids were detected using microscopy and PCR. Immunofluorescence analysis revealed knock-in in proximal tubule epithelial cells (LTL+). In knock-out experiments, live confocal microscopy indicated areas of GFP loss within kidney organoids treated with gRNA targeting GFP, but not with a scrambled guide. Mosaic patches of GFP knockout cells expanded over several days. Staining with nephron markers such as LTL and podocalyxin revealed knockout in both proximal tubule cells and podocytes. By next generation sequencing, the two-guide system produced larger deletions and was more efficient (20 % knockout), compared to single guide.


The strategy developed here is efficient for knocking in and knocking out genes in kidney epithelium. It uses commercially available reagents to perform CRISPR gene editing. sgRNA sequences or AAVS1 knock-in templates can be customized to target or introduce any gene of interest at specific loci. This provides a platform for the development of off-the-shelf gene therapies for diverse kidney disease states.


  • NIDDK Support