Rapid Genotyping of ApoL1 Risk Alleles using CRISPR-Cas12a

SUMMARY. African Americans are disproportionately affected by chronic and end stage renal disease (ESRD); while 35% of patients on dialysis are African American, only 13.2% of the U.S. population is African American. A major factor contributing to this disparity are genetic variations in apolipoprotein L1 (APOL1). APOL1 is a plasma protein protective against ‘African sleeping sickness’ caused by the parasite Trypanosoma brucei. There are three main allelic variants of APOL1: G0 (wild-type), G1, and G2. The G1 and G2 APOL1 alleles (i.e., renal risk alleles) impart resistance to sleeping sickness, while the G0 allele enables parasite survival and infection. For this reason, the G1 and G2 alleles are highly prevalent in individuals with African ancestry. The G1 and G2 variants of APOL1 are also present at relatively high frequencies among African Americans, with approximately 35% of the African American population having at least one G1 or G2 allele. Despite providing an advantage in survival from African trypanosomiasis, these genotypic variants predispose individuals to develop severe, irreparable kidney disease. People with two risk alleles, i.e., who are homozygous for either the G1 or G2 alleles or are doubly heterozygous for these alleles (G1/G2), have an APOL1 ‘risk genotype’ and are at elevated risk for developing focal segmental glomerulosclerosis (FSGS), which leads to progressive scarring and loss of function of glomeruli. Moreover, the risk genotype is associated with reduced allograft longevity in kidneys transplanted from donors with two risk alleles. More recently, it has been found that other glomerulopathies linked to viral infections, including HIV and SARS-CoV-2, are exacerbated by having the APOL1 risk genotype. Given that approximately 13% of African Americans have a genotype with two risk alleles, APOL1-linked kidney disease represents a potentially massive, yet still underappreciated, public health issue. The available methods for detecting pathological APOL1 variants, including gene sequencing and TaqMan, are relatively expensive and require specialized equipment and skills. Recently, CRISPR/Cas-based methods of detecting specific nucleic acid sequences have been developed. These methods are both simple and inexpensive and therefore offer significant advantages to conventional genotyping methods. In this Phase I application, we propose to develop a proof-of-concept CRISPR/Cas12a-based genotyping assay to detect the G0, G1, and G2 variants of ApoL1. Once developed and optimized, this assay will lead to a suite of reagents and techniques to expand access to simple and affordable ApoL1 genotyping that is less reliant on specialized equipment. Two novel therapeutic agents for treatment of APOL1-mediated kidney disease are currently in clinical trials, highlighting the urgency to develop better diagnostic tools that can identify individuals who could benefit from these treatments.PROJECT NARRATIVE. Approximately 13% of African Americans have a combination of pathological variants of the APOL1 gene, often termed the APOL1 “risk genotype,” that predisposes individuals to serious kidney diseases such as focal segmental glomerulosclerosis (FSGS). Despite the potential severity of APOL1-linked kidney disease, genetic testing for APOL1 variants is uncommonly performed, primarily due to the expense and lack of availability of testing. In this application, we propose to develop a CRISPR/Cas12a-mediated genotyping platform capable of rapid and inexpensive detection of pathological APOL1 variants from patient samples.