Development of a Non-Invasive CRISPR-Cas12a-LAMP Assay for Rapid Detection of ARHGAP21 and BARD1 Methylation Signatures in Nigerian Prostate Cancer Patients
Niels Nguedia Kaze, Irrinus Fonyuy Kintung, Aurelie Cailleau, Gilbert Fokou, Stephen Ikuerowo, Ogunlana O. Olubanke, Shalom ChineduPURPOSE
Prostate cancer (PCa) presents a severe mortality burden in Sub-Saharan Africa, especially in Nigeria, due to late clinical presentation, lack of population-specific markers for early detection, and the limitations of standard screening tools. To improve cancer management, understanding the population's genetic or epigenetic profile is vital for biomarker identification, reason why this study aimed to develop an affordable, non-invasive CRISPR-Cas12a-LAMP diagnostic assay designed to rapidly detect PCa-associated methylation signatures in caretaker genes ARHGAP21 and BARD1, easily deployable in low-resource settings.
METHODS
A case-control study was conducted, including 100 participants (40 histologically confirmed PCa cases, 30 with Benign Prostatic Hyperplasia [BPH], and 30 healthy volunteers) recruited from the Lagos State University Teaching Hospital. While blood and urine were collected, analytical optimization was performed exclusively on blood-derived DNA due to the extract quality. Promoter methylation of ARHGAP21 and BARD1 was characterized using Methylation-Sensitive Restriction Enzyme PCR (MSRE-PCR) with HpaII and MspI, coupled with cross-validated qPCR. Validated targets were subsequently used to design a CRISPR-Cas12a-LAMP assay via standard laboratory heating blocks and incubators.
RESULTS
Epigenetic profiling revealed that the BARD1 promoter was entirely unmethylated across the cohort. In contrast, ARHGAP21 promoter hypermethylation was exclusively identified in 20% of the PCa cases, confirmed by qPCR. The fluorescent CRISPR-Cas12a-LAMP assay successfully detected these ARHGAP21 methylation signatures within 50min reactions after DNA digestion, demonstrating a diagnostic sensitivity of 75% and a Positive Predictive Value (PPV) of 75% when compared to the qPCR standard.
CONCLUSION
We successfully developed a rapid fluorescent CRISPR-Cas12a-LAMP assay to detect methylation of the ARHGAP21 promoter accurately in the PCa group without requiring an expensive thermocycler. This provides a deployable molecular diagnostic tool for early PCa screening in resource-limited clinical environments.