Analytica chimica acta · 2026
CbAgo-enriched Cas12a biosensor for cancer mutations screening.
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Zhai Xiwen, Yang Biyao, Deng Fei, Gulati Sneha, Mckinnirey Flyn, Wu Xuan, Li Yi, Goldys Ewa M
Аннотация
BACKGROUND: Accurate detection of low-frequency DNA mutations in body fluids is essential for cancer monitoring and treatment evaluation. However, the high abundance of wild-type DNA often masks rare mutant signals, making sensitive detection particularly challenging.
RESULTS: We developed a screening strategy termed the CbAgo-enriched Cas12a mutation screening system (CECMS). By integrating the single-nucleotide resolution of CbAgo with the trans-cleavage activity of CRISPR-Cas12a, this system selectively eliminates wild-type DNA while enriching targeted mutant alleles. CECMS achieves up to 100-fold higher sensitivity at 37 °C compared with conventional Cas12a biosensors, enabling reliable detection of variant allele frequencies (VAFs) as low as 0.01%. In undiluted serum spiked samples for circulating tumor DNA (ctDNA) detection, the method successfully detected pancreatic cancer-associated KRAS G12D mutations at a VAF of 0.1%.
SIGNIFICANCE: By leveraging CbAgo-mediated enrichment, the capability of exposing rare SNV for downstream detection is markedly improved. With its high efficiency and ease of use, CECMS holds strong potential as a convenient tool for clinical cancer diagnostics and monitoring.
RESULTS: We developed a screening strategy termed the CbAgo-enriched Cas12a mutation screening system (CECMS). By integrating the single-nucleotide resolution of CbAgo with the trans-cleavage activity of CRISPR-Cas12a, this system selectively eliminates wild-type DNA while enriching targeted mutant alleles. CECMS achieves up to 100-fold higher sensitivity at 37 °C compared with conventional Cas12a biosensors, enabling reliable detection of variant allele frequencies (VAFs) as low as 0.01%. In undiluted serum spiked samples for circulating tumor DNA (ctDNA) detection, the method successfully detected pancreatic cancer-associated KRAS G12D mutations at a VAF of 0.1%.
SIGNIFICANCE: By leveraging CbAgo-mediated enrichment, the capability of exposing rare SNV for downstream detection is markedly improved. With its high efficiency and ease of use, CECMS holds strong potential as a convenient tool for clinical cancer diagnostics and monitoring.