Multiplexed Differential Gene Expression Analysis Using CRISPR-Cas Systems

Abstract

Quantification of mRNA expression levels and differential analyses that observe changes in mRNA expression are key in providing insights into biological processes. Today, the gold standard of differential gene expression analysis involves an initial genome-wide screen of expression changes using RNA-Seq, an expensive procedure that has a multiple-week turnaround time for results, and subsequent analyses of specific genes identified through RNA-Seq using the faster and less expensive technique of qPCR. While this workflow allows for effective analysis of changes in gene expression in small sets of key genes, it is too cumbersome for analyzing a larger set of genes or treatment conditions. This project sought to develop a highly multiplexed method of mRNA quantitation using CRISPR-Cas systems and microfluidics devices to parallelize thousands of detection reactions in a single experimental run. Core experimental components such as divalent metal cation concentrations and T7 promoter regions were systematically optimized to maximize the dynamic range of Cas-based quantitation and to improve the limits of quantitation. The developed system was used to quantify changes in transcript levels in yellow-fever-infected human hepatoma cells. These results were compared with gold-standard qPCR results and a strong correlation was observed between Cas-based quantitation and qPCR quantitation.