CRISPR-enabled point-of-care genotyping for APOL1 genetic risk assessment

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Genetic variant detection is crucial for personalized medicine, but current methods are not suitable for point-of-care (POC) settings.
• CRISPR-based diagnostics offer high sensitivity and specificity for POC applications, but their use in genotyping is underexplored.

Purpose of the Study:

• To develop a multiplexed CRISPR-based genotyping assay for simultaneous detection of multiple genetic variants.
• To apply the assay for identifying APOL1 gene variants associated with kidney disease risk in a clinical cohort.
• To demonstrate the feasibility of a simplified, POC-compatible readout for genetic analysis.

Main Methods:

• Utilized a multiplexed CRISPR system with LwaCas13a, PsmCas13b, and LbaCas12a nucleases.
• Developed a CRISPR-based assay for simultaneous detection of six genotypes.
• Applied machine learning for robust genotype analysis in over 100 patients.
• Integrated a multi-analyte lateral-flow assay for simplified readout.

Main Results:

• Successfully identified six genotypes simultaneously using the multiplexed CRISPR assay.
• Accurately determined APOL1 gene genotypes in a multicenter clinical cohort of over 100 patients.
• Demonstrated simplified genotype determination of clinical samples using a lateral-flow assay.

Conclusions:

• The developed CRISPR-based genotyping assay is sensitive, specific, and suitable for point-of-care applications.
• This technology enables cost-effective and rapid genetic variant detection, including for disease-associated variants like APOL1.
• The assay's versatility and simplified readout facilitate broader clinical utility for genetic screening and risk assessment.