High-Fidelity CRISPR-Cas12a–Based Apolipoprotein L1 Genotyping

Key Points A protospacer adjacent motif sequence is not required for CRISPR-Cas12a–mediated cis-cleavage of apolipoprotein L1 risk allele sequences. Discrimination of both single-nucleotide and insertion/deletion variants can be accurately targeted using short 17-nucleotide clustered regularly interspaced short palindromic repeats RNA guides. Both fluorescence-based and lateral flow assays can be used for apolipoprotein L1 genotyping with 100% sensitivity and specificity. Background Approximately 13% of Black people have a combination of risk variants of the apolipoprotein L1 ( APOL1 ) gene, often termed the APOL1 “high-risk genotype,” which predisposes individuals to serious kidney diseases, such as focal segmental glomerulosclerosis. Despite the potential severity of APOL1-linked kidney disease, genetic testing for APOL1 variants is uncommonly performed. Point-of-care testing has the potential to be transformative in patient care to immediately identify risk status, particularly for the evaluation of deceased donor organs in the setting of transplantation, but no assays are available to date. Methods To begin to address this unmet need, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a–mediated genotyping assay for the detection of pathological APOL1 variants from patient samples for potential use in a point-of-care setting. For each variant, clustered regularly interspaced short palindromic repeats RNA guides were identified that could readily discriminate between either the wild-type or variant sequence in a Cas12a- trans -cleavage assay using both fluorescence- and lateral flow–based detection methods. Results Interestingly, no protospacer adjacent motif sequence was required for the CRISPR-Cas12a–based detection of any of the variants. CRISPR-Cas12a fluorescence-based genotyping assays performed on patient samples achieved 100% sensitivity and specificity compared with genotypes obtained using a clinically validated TaqMan assay. Similar results were also obtained in the lateral flow assay format with a 6-carboxyfluorescein- and biotin-labeled reporter. Conclusions The successful deployment in this study of Cas12a-based assays to generate APOL1 genotypes for individuals with high fidelity provides promise for a genotyping method potentially amenable to point-of-care use.