Multiple CRISPR zones-driven ultrasensitive detection of DNA via CRISPR-Cas12a and ligation-rolling circle amplification.

The ability to detect specific DNA, including single nucleotide variants (SNVs), with high sensitivity is essential for advancing genetic research, diagnostics, and personalized medicine. This study presents a novel method for ultrasensitive DNA detection, combining ligation-rolling circle amplification (L-RCA) with CRISPR-Cas12a. While L-RCA systems have been widely used for nucleic acid detection, the sensitivity of conventional L-RCA generally reaches approximately 100 pM. Here, we demonstrate that the sensitivity of RCA-Cas12a systems can be markedly enhanced by incorporating multiple CRISPR target regions into the padlock probe. This method achieves remarkable sensitivity, detecting DNA at concentrations as low as 1 aM (6 copies per reaction), and is capable of identifying single nucleotide variants (SNVs) with allele fractions as low as 1 %. Unlike many current complex RCA-Cas12a strategies, this approach is simple and does not require advanced labeling or instrumentation, making it a promising tool for ultrasensitive DNA detection in various applications.