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Published on: December 23, 2022
CbAgo-enriched Cas12a biosensor for cancer mutations screening
Xiwen Zhai1, Biyao Yang1, Fei Deng2
1School of Biomedical Engineering, ARC Centre of Excellence in Nanoscale Biophotonics, Faculty of Engineering, UNSW Sydney, NSW, 2052, Australia.
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.
