CRISPR-Cas systems for diagnosing infectious diseases

Abstract

Infectious diseases are a global health problem affecting billions of people. Developing rapid and sensitive diagnostic tools is key for successful patient management and curbing disease spread. Currently available diagnostics are very specific and sensitive but time-consuming and require expensive laboratory settings and well-trained personnel; thus, they are not available in resource-limited areas, for the purposes of large-scale screenings and in case of outbreaks and epidemics. Developing new, rapid, and affordable point-of-care diagnostic assays is urgently needed. This review focuses on CRISPR-based technologies and their perspectives to become platforms for point-of-care nucleic acid detection methods and as deployable diagnostic platforms that could help to identify and curb outbreaks and emerging epidemics. We describe the mechanisms and function of different classes and types of CRISPR-Cas systems, including pros and cons for developing molecular diagnostic tests and applications of each type to detect a wide range of infectious agents. Many Cas proteins (Cas3, Cas9, Cas12, Cas13, Cas14 etc.) have been leveraged to create highly accurate and sensitive diagnostic tools combined with technologies of signal amplification and fluorescent, potentiometric, colorimetric, lateral flow assay detection and other. In particular, the most advanced platforms -- SHERLOCK/v2, DETECTR, CARMEN or CRISPR-Chip -- enable detection of attomolar amounts of pathogenic nucleic acids with specificity comparable to that of PCR but with minimal technical settings. Further developing CRISPR-based diagnostic tools promises to dramatically transform molecular diagnostics, making them easily affordable and accessible virtually anywhere in the world. The burden of socially significant diseases, frequent outbreaks, recent epidemics (MERS, SARS and the ongoing COVID-19) and outbreaks of zoonotic viruses (African Swine Fever Virus etc.) urgently need the developing and distribution of express-diagnostic tools. Recently devised CRISPR-technologies represent the unprecedented opportunity to reshape epidemiological surveillance and molecular diagnostics.

Keywords: COVID-19; HBV; HIV; HPV; Mobile phone microscopy; Molecular diagnostics; Molecular epidemiology; One pot assays; Point-of-care (POC); SARS-CoV-2; Tuberculosis; Viruses.

Fig. 1

Schematics of CRISPR-Cas9-based CRISPR-diagnostic method CASLFA. (A) Structure of the lateral flow device. The lateral flow device consists of a sample pad where the isolate is applied, a conjugate pad with pre-assembled AuNP-DNA probes, a test line and a control line. At the test line, complexes of CRISPR-Cas with the target biotinylated DNA and AuNP-DNA probes, hybridized with the stem-loop region of sgRNA, interact with pre-coated streptavidin at the test pad to produce a visible signal. At the same time, AuNP-DNA probes move further and interact with streptavidin at the control line. AuNP-DNA probes contain three regions, namely (1) polyA-polyT (poly A used for labeling with Au and polyT as a linker); (2) purple area for hybridization with the embedded probe in the control line and (3) yellow area used for hybridization with the engineered stem-loop region in sgRNA. (B) Schematics of CASLFA procedure. Isolated DNA is amplified with biotinylated primers using RPA or PCR. Amplicons are mixed with CRISPR-Cas9 detection complex and DNA probes and, after short incubation, applied to the lateral flow device. The picture was created in BioRender. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Schematics of CRISPR-Cas DETECTR and OR-DETECTR diagnostic platform. (A) DETECTR pipeline. The DNA molecule is amplified using isothermal amplification RPA method followed by the addition of the Cas12a mix with sgRNA and fluorescent probes. Cas12a recognizes the target DNA and destroyes fluorescent probes by means of collateral activity to produce a fluorescent signal. (B) OR-DETECTR pipeline. CRISPR-Cas mix and RT-RPA mix are physically separated to avoid opening the tube and potential cross-contamination of the samples. The picture was created in BioRender.

Fig. 3

Schematics of CRISPR-Cas diagnostic platforms SHERLOCK/v2. SHERLOCK/SHERLOCKv2 pipeline. DNA or RNA molecules are isothermally amplified using RPA or RT-RPA, correspondingly. DNA is transcribed into RNA using in vitro T7 transcription reaction. Cas13 recognizes target RNA molecules and cleaves fluorescent probes by means of collateral activity. Different nucleotide preferences of Cas13 proteins from different species can be used for preferential cleavage of fluorescent probes at specific dinucleotides. Thus, this method can be used for multiplex detection with designed probes. Alternatively, reaction results can be visualized on lateral flow strips using chromogenic reaction. The picture was created in BioRender.

Fig. 4

Pipeline and principle of ultralocalized Cas13a assay. (A) Schematics of ultralocalized Cas13a assay. Pico-sized droplets are mixed with target RNA and CRISPR-Cas13a detection reaction. Positive signal results in illumination of the droplets that can be counted by fluorescent microscope. (B) The principle of confinement effect on local concentration of target molecules. Upon decrease in the analytical volume, the local concentration of target molecules inversely increases. The picture was created in BioRender.

Fig. 5

Schematics of CARMEN-Cas13 detection method. (1) amplification of target nucleic acids and their emulsification with color codes. (2) Generation of emulsions with CRISPR-Cas detection systems. (3) Pooling of two mixes. (4) Loading of mixed emulsions into the chip. (5) Schematic representation of a droplet with target molecules (sample droplet) and a droplet with CRISPR/Cas detection mix (CRISPR detection droplet). After merging, CRISPR-Cas system interacts with the target molecules, producing a specific fluorescent signa. The picture was created in BioRender.