Abstract: SA-PO762
A Whole-Genome CRISPR Knockout Positive Screen Reveals Pathways Regulating APOL1-Induced Cytotoxicity
Session Information
- CKD: Mechanisms - III
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 2103 CKD (Non-Dialysis): Mechanisms
Authors
- Oltrabella, Francesca, Genentech, South San Francisco, California, United States
- Lau, Ted, Genentech, South San Francisco, California, United States
- Cooper, Jonathan, Genentech, South San Francisco, California, United States
- Callow, Marinella, Genentech, South San Francisco, California, United States
- Haley, Benjamin, Genentech, South San Francisco, California, United States
- Martin, Scott, Genentech, South San Francisco, California, United States
- Costa, Michael, Genentech, South San Francisco, California, United States
- Scales, Suzie J., Genentech, South San Francisco, California, United States
Background
In humans two genetic variants of Apolipoprotein L1 (APOL1), G1 and G2, are common in African Americans and are strongly associated with chronic kidney disease (CKD). However, little is known about their pathological mechanism(s). APOL1 G1 and G2 are generally more toxic than wild type upon overexpression, with evidence for cellular mechanisms ranging from mitochondrial dysfunction, ER stress, endosome or autophagosome maturation defects, impaired cholesterol efflux, altered suPAR:integrin binding to cation efflux. It is unlikely that all these are primarily responsible for CKD, so an unbiased approach to identifying pathways and/or co-factors that mediate APOL1 variant cell killing should aid our understanding of the actual disease mechanism.
Methods
A pooled genome-wide CRISPR knockout screen, where each cell is deleted of a single gene prior to a phenotype-based selection, is one such powerful unbiased technique, which APOL1-induced in vitro cell death renders possible. Here, we show how we performed a genome-scale CRISPR-Cas9 loss-of-function screen in HEK293 cells to discover genes that influence APOL1-induced cell killing. We generated Tet inducible APOL1 G0, G1 and G2 HEK-293 cells expressing Cas9-GFP and carefully titrated APOL1 variant dose-dependent cell killing. These cells were infected with a genome-wide lentiviral guide RNA library, followed by APOL1 induction to the pre-determined level.
Results
The cells surviving APOL1 selective pressure were subjected to next-generation sequencing of the integrated sgRNA cassettes. With this approach we identified new and existing pathways affecting APOL1-mediated cell killing.
Conclusion
Here we demonstrated how APOL1 variants over-expression could be used as a selective pressure in a CRISPR KO positive screen to reveal pathways that are directly or indirectly involved in APOL1-induced cell toxicity. The same pathways have the potential to become druggable targets for patients that develop CKD linked to APOL1 variants. Validation and characterization analysis of the discovered factors will be essential to confirm our findings and to demonstrate the feasibility of our screening approach.
Funding
- Commercial Support –