Project Description

Abstract

MicroRNAs, serve as critical gene expression regulators at the transcriptional and post-transcriptional levels, have also been found as important blood-based biomarkers for early detection of cancers. However, their current in vitro detection methods are relatively complex, costly and low sensitive. Our project attempts to establish a novel in vitro microRNA detection system which is rapid, efficient, sensitive and specific. In this system, CRISPR-Cas9 technique is modified to integrate with split-luciferase or split-HRP reporting systems. The advanced rolling circle amplification technology and cell-free expression system are also involved and optimized. This system may ideally be compatible for the detection of various series of small non-coding RNAs. To our knowledge, we are the first to use the CRISPR-Cas9 system as a small non-coding RNA monitor in vitro. Its establishment and further development might provide a new approach for rapid and low-cost cancer screening, virus detection and curative efficacy assessment.

Introduction

Nowadays, cancers, due to their high incidence and serious mortality, are affecting populations in all countries and all regions (Figure 1). However, in most countries, resources for prevention and diagnosis of cancer still remain limited due to their high cost and low cost-effectiveness¹, whereas the early detection of cancer has been proven to result in improved survival, less extensive treatment and less possibility to metastasis^2-4. Such situation highlighted the undiminished importance of the development of a low-cost, easily accessible and rapid tool for early screening and detection of cancers.

MicroRNAs (miRNAs), as a kind of small non-coding RNA containing approximately 22 nucleic acids, have been proven to play important roles on post-transcriptional regulation of the gene expression, thus involving in the regulation of many important biological events⁵. Recently, it was reported that serum miRNAs can serve as a promising cancer biomarker because their expression pattern can be correlated with cancer type, stage, and other clinical variables, which then, implying that miRNA profiling can be used as a tool for cancer diagnosis and prognosis^6-8. Moreover, circulating miRNAs have been proven to remain stable under some extreme condition such as RNase exposure, multiple freeze-thaw cycles, and extreme pH, thus making them strong candidates for low-cost detection and analysis ⁹. However, due to their short length, low expression level and high homologous sequence similarity, the quantified detection and analyzation of circulating miRNAs remain challenging nowadays. Old-schools such as Northern Blotting, microarray and qRT-PCR technique are still our approach to detect and analyze the quantity of miRNA¹⁰. Notably, the expanded application of these techniques, as well as some other new approaches such as bioluminescence¹¹, Nanopore sensors¹² were severely limited due to their relatively low sensitivity (which were mostly nM sensitivity against the pM or even fM concentration of blood miRNA), cumbersome and complex in operation, and relative high cost. More recently, Deng et.al reported a single-molecule resolution in situ miRNA detection technique based on rolling circle amplification (RCA)¹³ . However, this approach has been restricted only in the application of cellular in situ analysis. Its expandability to circulating miRNA detection still faces a major problem that the degree of one-step signal amplification and differentiation might not be sufficient to meet the requirements of sensitivity and specificity. At the main time, such method still relies on equipment such as Fluoresce microplate readers or fluorescence microscopes, which are highly costly.