Engineering CRISPR/Cas12 Components Toward Simple and Robust Next-Generation Diagnostics

Type V and VI CRISPR/Cas systems have emerged as promising cutting-edge diagnostic tools for nucleic acid detection. Nevertheless, the challenge of low target concentrations necessitates pre-amplification, which constrains their practical use in point-of-care (POC) settings. Our lab has explored multiple strategies to create a platform for detecting nucleic acids that is as quick and simple as rapid antigen tests but has an accuracy of a qPCR test.

Firstly, in pursuit of a unified testing method that combines RT-LAMP and CRISPR at a single operating temperature, we delved into various thermophilic Cas12b orthologs. This exploration led to the discovery of BrCas12b, which possessed exceptional thermostability and enabled the development of a one-pot assay known as SPADE. SPADE successfully identified 5 major SARS-CoV-2 variants at temperatures between 60-62°C. Secondly, employing a combination of structural design and machine learning techniques, we engineered BrCas12b to further enhance its thermophilicity, resulting in the creation of a robust one-pot assay named SPLENDID. The SPLENDID effectively operates within the desirable temperature range of 65-67°C and demonstrates remarkable accuracy in detecting HCV genotype 1 and SARS-CoV-2 RNA clinically.

Thirdly, our research unveiled various CRISPR/Cas12a orthologs capable of partially recognizing native RNAs. This discovery paved the way for the development of SAHARA, a split activator system designed for target RNA detection using CRISPR/Cas12a without the need for reverse transcription. SAHARA represents an amplification-free and reverse-transcription-free one-pot detection technology for the direct analysis of RNAs. Furthermore, our laboratory has been actively investigating methods to develop PAM-less approaches for detecting nucleic acids and single-point mismatches without constraints. These groundbreaking advancements hold the potential to revolutionize the next generation of diagnostic tools, effectively addressing the pressing need for rapid, accurate, and accessible point-of-care tests for infectious diseases.

Learning Objectives: 

1. Recognize the need for a simple and accurate point-of-care test for diagnosing infectious diseases, genetic disorders, and beyond.

2. Identify the limitations of existing platform detection technologies in terms of simplicity, speed, sensitivity, specificity, and stability.

3. Describe how CRISPR/Cas systems can be engineered to develop a simple and robust next-generation point-of-care test for any genetic target.