Target-induced transcription amplification to trigger the trans-cleavage activity of CRISPR/Cas13a (TITAC-Cas) for detection of alkaline phosphatase

Abstract

Herein, an ultra-sensitive alkaline phosphatase (ALP) sensing strategy is developed by target-induced transcription amplification to trigger the trans-cleavage activity of Cas13a (TITAC-Cas). A double-stranded DNA duplex integrating a T7 promoter with 5′-phosphate and a transcription template (5′P-dsDNA) serves as the ALP substrate. In the absence of ALP, 5′P-dsDNA can be degraded by the λexo, leading to the subsequent transcription failure. In the presence of ALP, dephosphorylation reaction converts the 5′P-dsDNA to 5′OH-dsDNA and provides the protection for T7 promoter against the λexo-digestion. The intact T7 promoter of 5′OH-dsDNA can activate T7 transcription to produce a mass of single-stranded RNA (ssRNA). The ssRNA products possess a full complementarity to the spacer of crRNA and activate the ssRNase activity of CRISPR/Cas13a. As a result, Cas13a exhibits the indiscriminate cleavage of collateral FQ-reporter to release significant fluorescence signal, realizing the ultra-sensitive detection of ALP. Due to the triple signal amplification (ALP self-catalysis, T7 transcription amplification, and trans-cleavage of CRISPR/Cas13a), TITAC-Cas assay shows the ultra-sensitive detection of ALP activity with a wide linear range from 0.008 to 250 U∙L−1). The LOD is calculated to be 6 ± 0.52 mU∙L−1. TITAC-Cas assay is also successfully applied for analysis of ALP activity in HepG2 cell lysate with high fidelity. In addition, this method is employed to screen ALP inhibitor.