One-tube target amplification-free CRISPR-Cas13 autocatalytic system

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) diagnostics have shown great promise for nucleic acid testing. However, CRISPR/Cas13a-based RNA sensing is hindered by the absence of accurate, fast and sensitive assays. Current Cas-based methods generally involve two separate steps: target-amplification and Cas-detection. This two-step method complicates detection, causes cross-contamination, and is difficult to deploy. Therefore, it is necessary to develop a facile one-step Cas13a-based RNA detection assay. We present a one-tube target amplification-free CRISPR-Cas13a autocatalytic circuit system that combines the Cas13a nuclease, T4 polynucleotide kinase, Bsu DNA polymerase and T7 RNA polymerase with a universally designed combinatorial DNA probe to create a one-pot assay for isothermal RNA detection. In this assay, after target RNA recognition, the cis-cleavage and trans-cleavage activities of Cas13 are activated, which leads to the initiation of a Cas13a autocatalytic reaction, resulting in an exponentially amplifying detection signal, thus enabling the detection of approximately 23.01 (95 % CI:16.95-41.23) copies/µL RNA in 45 min. A facile, easy-to-deploy, and one-tube Cas13a-based autocatalytic reaction system was constructed for rapid and highly sensitive RNA detection for the first time. This Cas13a-based autocatalytic system offers not only a versatile platform for directly detecting RNA targets but also a route toward point-of-care nucleic acid testing.