Development of CRISPR-Mediated Nucleic Acid Detection Technologies and Their Applications in the Livestock Industry

Abstract

The rapid rate of virus transmission and pathogen mutation and evolution highlight the necessity for innovative approaches to the diagnosis and prevention of infectious diseases. Traditional technologies for pathogen detection, mostly PCR-based, involve costly/advanced equipment and skilled personnel and are therefore not feasible in resource-limited areas. Over the years, many promising methods based on clustered regularly interspaced short palindromic repeats and the associated protein systems (CRISPR/Cas), i.e., orthologues of Cas9, Cas12, Cas13 and Cas14, have been reported for nucleic acid detection. CRISPR/Cas effectors can provide one-tube reaction systems, amplification-free strategies, simultaneous multiplex pathogen detection, visual colorimetric detection, and quantitative identification as alternatives to quantitative PCR (qPCR). This review summarizes the current development of CRISPR/Cas-mediated molecular diagnostics, as well as their design software and readout methods, highlighting technical improvements for integrating CRISPR/Cas technologies into on-site applications. It further highlights recent applications of CRISPR/Cas-based nucleic acid detection in livestock industry, including emerging infectious diseases, authenticity and composition of meat/milk products, as well as sex determination of early embryos.

Multiplex Detection of Infectious Diseases on Microfluidic Platforms

“…PCR is typically considered as the gold standard detection method of nucleic acid-based diagnosis, but involves costly/advanced equipment and skilled personnel, so it cannot be easily combined with microfluidic technology [ 53 ]. Therefore, isothermal amplification and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR/Cas) system, which have the advantage of low-cost, reliability, and do not require for bulky equipment, can be compatible with the microfluidic platforms for nucleic acid detection [ 20 , 54 ]. Based on the spatial separation of detection sites on microfluidic platforms, the multiplex detection of different nucleic acids can be realized [ 14 ].…”