CRISPR-Cas-Integrated LAMP

Abstract

Pathogen-specific point-of-care (PoC) diagnostic tests have become an important need in the fight against infectious diseases and epidemics in recent years. PoC diagnostic tests are designed with the following parameters in mind: rapidity, accuracy, sensitivity, specificity, and ease of use. Molecular techniques are the gold standard for pathogen detection due to their accuracy and specificity. There are various limitations in adapting molecular diagnostic methods to PoC diagnostic tests. Efforts to overcome limitations are focused on the development of integrated molecular diagnostics by utilizing the latest technologies available to create the most successful PoC diagnostic platforms. With this point of view, a new generation technology was developed by combining loop-mediated isothermal amplification (LAMP) technology with clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) technology. This integrated approach benefits from the properties of LAMP technology, namely its high efficiency, short turnaround time, and the lack of need for a complex device. It also makes use of the programmable function of CRISPR-Cas technology and the collateral cleavage activity of certain Cas proteins that allow for convenient reporter detection. Thus, this combined technology enables the development of PoC diagnostic tests with high sensitivity, specificity, and ease of use without the need for complicated devices. In this review, we discuss the advantages and limitations of the CRISPR/Cas combined LAMP technology. We review current limitations to convert CRISPR combined LAMP into pathogen-specific PoC platforms. Furthermore, we point out the need to design more useful PoC platforms using microfabrication technologies by developing strategies that overcome the limitations of this new technology, reduce its complexity, and reduce the risk of contamination.

Keywords: CRISPR/Cas combined LAMP technology; clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas); loop-mediated isothermal amplification (LAMP); point-of-care (PoC) platform.

Figure 1

Representation of the LAMP amplification principle in the schematic diagram. Reprinted from Ref. [14].

Figure 2

CRISPR/Cas technology. (A) Experimental process of CRISPR/Cas integrated LAMP technology [41]. (B) Schematic representation of the CRISPR/Cas combined LAMP diagnostic tests developed for the detection of SARS-CoV-2 [41]. Reprinted from Ref. [41].

Figure 3

Schematic representation of the experimental process of CRISPR/Cas12a integrated RT-LAMP technique and portable PoC device [51]. Reprinted from Ref. [51].