Kidney Week

Abstract: FR-PO0108

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Screening for Modulators of APOL1 G2-Induced Cytotoxicity

Session Information

• Hereditary Glomerular and Tubulointerstitial Kidney Diseases
  October 23, 2026 | Location: Exhibit Hall A, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1202 Genetic Diseases of the Kidneys: Non-Cystic (Complex and Non-Cystic Monogenic)

Authors

• Thomson, Russell Paul, Regeneron Pharmaceuticals Inc, Tarrytown, New York, United States

Russell Paul Thomson, PhD

• Dragileva, Ella, Regeneron Pharmaceuticals Inc, Tarrytown, New York, United States

Ella Dragileva

• Kissner, Kathryn L., Regeneron Pharmaceuticals Inc, Tarrytown, New York, United States

Kathryn L. Kissner

• Fury, Wen, Regeneron Pharmaceuticals Inc, Tarrytown, New York, United States

Wen Fury

• Rogulin, Vitaliy, Regeneron Pharmaceuticals Inc, Tarrytown, New York, United States

Vitaliy Rogulin

• Prissette, Marine, Regeneron Pharmaceuticals Inc, Tarrytown, New York, United States

Marine Prissette

• Zambrowicz, Brian P., Regeneron Pharmaceuticals Inc, Tarrytown, New York, United States

Brian P. Zambrowicz, PhD

Background

Common coding variants in the APOL1 gene, known as kidney-risk variants (KRVs), are associated with an increased risk of kidney disease among individuals of African descent. Kidney disease onset appears to result from increased APOL1 KRV expression in podocytes following virus-induced interferon production. Compared to the ancestral APOL1 genotype, known as G0, APOL1 KRVs, called G1 and G2, are cytotoxic when produced in human cells, and cause kidney disease when produced in APOL1 transgenic mice. The exact mechanism of cytotoxicity caused by APOL1 KRVs remains uncertain, although recent findings point to APOL1 ion channel activity at the plasma membrane and/or mitochondrial dysfunction. Here, we describe an unbiased genome-wide CRISPR KO screen to identify genetic modulators of APOL1 G2-induced cytotoxicity in HEK293 cells.

Methods

HEK293 APOL1^KO cells were modified with Cas9 and Dox-inducible APOL1 G0 or APOL1 G2. Dox-induced expression of APOL1 G2, but not APOL1 G0, resulted in cell death. APOL1^KO, APOL1 G0 and APOL1 G2 cell lines were then transduced with a genome-wide CRISPR KO library (Brunello; 76,000 gRNAs). To simulate the disease condition, cells were primed with type-I & II interferons and then treated with doxycycline over 14 days to induce APOL1 expression.

Results

Samples were taken for NGS before, and after dox-induction. A total of 1400 sgRNAs were identified as significantly enriched or depleted in the APOL1 G2 population compared to APOL1 G0 and APOL1^KO. These sgRNAs were assembled into a mini-library and subjected to secondary screening. Several sgRNA hits were confirmed to reduce APOL1 cytotoxicity in a low throughput assay. These protective sgRNAs were found to reduce cell surface levels of APOL1 protein and/or expression of APOL1 G2 mRNA.

Conclusion

Genome-wide CRISPR screening in HEK293 cells was used to identify targets of interest that modulate APOL1 KRV-induced cytotoxicity. Efforts are now underway to screen in human iPSC-derived podocytes expressing the APOL1 G2 variant from the endogenous locus.

Funding

• Commercial Support – Regeneron Pharmaceuticals