Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C034

Oral Communications

Using CRISPR technology to generate models of polycystic kidney disease

N. Perretta-Tejedor1, E. Rovira Barreira1, M. Seda2, D. Jenkins2, A. Woolf3, P. Winyard1, D. A. Long1

1. Developmental Biology and Cancer, UCL Great Ormond Street Institute of Child Health, London, United Kingdom. 2. Genetics and Genomic Medicine, UCL Great Ormond Street Institute of Child Health, London, United Kingdom. 3. Division of Cell Matrix Biology & Regenerative Medicine, University of Manchester, Manchester, United Kingdom.


Polycystic kidney diseases (PKD) are characterised by the formation and progressive growth of renal cysts. Autosomal dominant (AD)PKD, the commonest PKD, affects 1 in 1,000 individuals and is caused by mutations in either PKD1 (85 %) or PKD2 (15%), which encode for polycystin-1 and -2 respectively. These two proteins regulate proliferation, fluid secretion, ciliary function, cell-cell adhesion, and cell-matrix interaction of renal epithelial cells. Mutations of either PKD1 or PKD2 result in increased cyclic AMP signalling which promotes cyst formation. Understanding the molecular basis of PKD is essential for developing more effective therapies for patients and this requires clinically-relevant cellular models. To do this, we employed the CRISPR-Cas9 system to generate mutations in exon 36 of PKD1 in human embryonic kidney (HEK) 293 cells. Two different cell lines were generated: one with a homozygous mutation in PKD1 (c.10744delCC) and another with a compound heterozygous mutation (allele 1 c.10744delCC and allele 2 c.10732delCGAGCTTCCC), both of which led to a frameshift deletion in PKD1. Next, the cells were embedded in Matrigel and compared with HEK293 wild-type cells in 3-dimensional culture. Over a period of five days, seeded single cells of both wild-type and PKD1 genotypes proliferated to form discrete cell cultures, some containing apparent lumens. Examination of the size of 180 clusters found that those containing wild-type cells were significantly smaller than those composed of either homozygous and compound heterozygous PKD1 knock-down cells (p<0.05 in both cases). Next, we exposed the cells to forskolin, a cyclic AMP agonist. In wild-type cells, forskolin significantly increased the area of the clusters (p<0.05). In contrast, in both of the PKD1 knock-down cells, forskolin did not cause any significant change in the cluster size. In conclusion, we have generated a cellular model of ADPKD using CRISPR-Cas9 technology which may be used to test the effect of different drugs in cystogenesis or examine different biological process involved in ADPKD such as cell proliferation or migration.

Where applicable, experiments conform with Society ethical requirements