A quick and inexpensive method to detect cancer early by exponentially amplifying rare and scarce cancer mutations.

Early cancer detection is challenging due to the low levels of cancer-indicating mutant DNA compared to healthy, wild-type DNA. The background noise from the substantial amounts of healthy DNA overwhelms mutant signals. Some current approaches include - amplification refractory mutation system (ARMS)-PCR; next generation sequencing (NGS); CRISPR-mediated Ultra-sensitive detection of Target DNA (CUT)-PCR; and dynamic allele-specific hybridization (DASH). These approaches are often unable to enrich the mutant DNA to a high enough level for accurate detection. Furthermore, they are costly and time-intensive, making them impractical for a real-time point-of-care detection setting.

Haim H. Bau and his team combined two technologies, CRISPR-Cas9 and isothermal recombinase polymerase amplification (RPA), to develop the Programmable Enzyme-Assisted Selective Exponential Amplification (PASEA) assay. The exponential increase in mutant DNA fraction (MAF) that is achieved compared to the current methods allows for much easier early cancer detection in a shorter period of time.

PASEA is inspired by Darwin’s “survival of the fittest” wherein the scarce cancerous mutant DNA fraction with the superior trait is amplified exponentially and quickly dominates the landscape. The wild-type DNA are also amplified, but at a much slower rate since they are detected and cleaved via CRISPR-Cas9. In just 20 minutes, the initially scant mutant DNA can be amplified from 0.01% up to 70% mutant DNA fraction (via RPA), followed by inexpensive Sanger sequencing to determine the genetic sequence of the mutant DNA. The assay can be applied to a microfluidic chip for point-of-care detection if desired.

• In 20 min, PASEA enriches MAF 3-222x better than DASH and CUT-PCR (in 60 min) depending on MAF % before enrichment outlined in table: 

 

• PASEA is three times faster than DASH and CUT-PCR for incubation times (20 min vs. 60 min)
• Can be implemented in real time point-of-care setting
• PASEA resulted in same sensitivity and accuracy as ARMS-PCR combined with NGS but lower cost and less time