Ligation-assisted DNA strand displacements and CRISPR/Cas14 cleavage for ultrasensitive detection of PIWI-interacting RNA.

PIWI-interacting RNA (piRNA) as a type of small non-coding RNA holds great promise for managing disease occurrence and progression. Herein, we construct a ligation-assisted DNA strand displacement strategy for enhanced CRISPR/Cas14 cleavage, which provides remarkable SERS and electrochemical responses to indicate trace piRNA information. The upstream reaction is rationally designed to integrate traditional strand displacement amplification (SDA) and ligation-mediated target recycling, which benefits improvement of the efficiency. The generated single-stranded DNA products can activate trans-cleavage activity of Cas14, enabling the DNA structural changes on the sensing interface and subsequent signal measurements. By coupling ligation-assisted SDA (LSDA) and CRISPR/Cas14 system, ultrahigh sensitivity is achieved with low background signal. This method not only reports consistent results comparing with standard qRT-PCR technique, but can discriminates piRNA expressions in samples from breast cancer patients. Therefore, a reliable and practical tool is provided for piRNA related clinical applications.