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Updated: May 22, 2025
Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a
Published on: December 23, 2022
Multiple CRISPR zones-driven ultrasensitive detection of DNA via CRISPR-Cas12a and ligation-rolling circle
Guigao Lin1, Jing Li2, Kuo Zhang1
1National Center for Clinical Laboratories, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/ National Center of Gerontology, PR China; Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, PR China; Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, PR China.
Abstract:
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.
