CRISPR‑based diagnostic approaches: Implications for rapid management of future pandemics (Review)

Abstract

Sudden viral outbreaks have increased in the early part of the 21st century, such as those of severe acute respiratory syndrome coronavirus (SARS‑CoV), Middle East respiratory syndrome corona virus, and SARS‑CoV‑2, owing to increased human access to wildlife habitats. Therefore, the likelihood of zoonotic transmission of human‑associated viruses has increased. The emergence of severe acute respiratory syndrome coronavirus 2 in China and its spread worldwide within months have highlighted the need to be ready with advanced diagnostic and antiviral approaches to treat newly emerging diseases with minimal harm to human health. The gold‑standard molecular diagnostic approaches currently used are time‑consuming, require trained personnel and sophisticated equipment, and therefore cannot be used as point‑of‑care devices for widespread monitoring and surveillance. Clustered regularly interspaced short palindromic repeats (CRISPR)‑associated (Cas) systems are widespread and have been reported in bacteria, archaea and bacteriophages. CRISPR‑Cas systems are organized into CRISPR arrays and adjacent Cas proteins. The detection and in‑depth biochemical characterization of class 2 type V and VI CRISPR‑Cas systems and orthologous proteins such as Cas12 and Cas13 have led to the development of CRISPR‑based diagnostic approaches, which have been used to detect viral diseases and distinguish between serotypes and subtypes. CRISPR‑based diagnostic approaches detect human single nucleotide polymorphisms in samples from patients with cancer and are used as antiviral agents to detect and destroy viruses that contain RNA as a genome. CRISPR‑based diagnostic approaches are likely to improve disease detection methods in the 21st century owing to their ease of development, low cost, reduced turnaround time, multiplexing and ease of deployment. The present review discusses the biochemical properties of Cas12 and Cas13 orthologs in viral disease detection and other applications. The present review expands the scope of CRISPR‑based diagnostic approaches to detect diseases and fight viruses as antivirals.

Keywords: CRISPR; CRISPR RNA; CRISPR‑associated systems; collateral activity; diagnostics; fluorescence‑quencher; point‑of‑care devices; reporter; sensitivity; specificity.

Figure 1.

Cas12 and Cas13 Cas orthologs recognize dsDNA and ssRNA possessing ssDNA and ssRNA cleaving trans-collateral activity. (A) CRISPR-Cas systems can be divided into two classes and six types. The class II system encodes single subunit enzymes, such as Cas9, Cas12 and Cas13, that target nucleic acids for modifications. Schematic representations of (B) Cas9, (C) Cas12 and (D) Cas13. Cas9 contains three RuvC (I, II and III) and one catalytically active HNH domain required to induce DNA cleavage. Cas12 contains three RuvC (I, II and III) endonuclease domains. Cas13 has two HEPN-binding domains, as presented in the figure. (E) Cas9 is presented in complex with sgRNA and target DNA. The position of PAM, target binding and cleavage position are marked in the figure. (F) Cas12 is presented in complex with crRNA and a double-stranded DNA target. The position of PAM and the cleavage sites are also presented. Cas12 also possesses promiscuous ssDNA degrading activity. (G) Cas13 is presented in complex with crRNA and ssRNA targets. Cas13 also possesses ssRNA-degrading collateral activity when complexed with target RNA. (H) The crRNA structure demonstrated in type VI b-Cas orthologs. The pre-crRNA processing occurs at the 3′ ends, which create spacers. (I) The most common crRNA structure reported in type a/c/d Cas orthologs in which processing of pre-crRNA occurs at the 5′ end, creating spacers. This figure was generated using BioRender (www.biorender.com). CRISPR, clustered regularly interspaced short palindromic repeats; Cas, CRISPR-associated; PAM, protospacer-associated motif; Indels, insertions or deletions; ss, single-stranded; sgRNA, single guide RNA; crRNA, CRISPR RNA; HEPN, higher eukaryotic and prokaryotic nucleotide; RuvC, an endonuclease domain named for an Escherichia coli protein involved in DNA repair; HNH, an endonuclease domain with catalytic histidine and asparagine residues.

Figure 2.

The discovery of Cas12 and Cas13 Cas orthologs has revolutionized the development of CRISPR-based diagnostics. Timeline of the major discoveries for CRISPR-Cas systems in nucleic acid detection. The discovery of CRISPR-Cas systems in bacteria and archaea, together with biochemical characterization of Cas12 and Cas13 ortholog enzymatic properties, has revolutionized the development of CRISPR-based diagnostics. The nonspecific trans-collateral activity of Cas12 and Cas13 in cleaving single-stranded DNA and single-stranded RNA has been exploited in the design and development of CRISPR-based diagnostic approaches for nucleic acid detection. CRISPR, clustered regularly interspaced short palindromic repeats; Cas, CRISPR-associated; SHERLOCK, specific high-sensitivity enzymatic reporter unlocking; v.1, version 1; HOLMES, 1-h low-cost multipurpose highly efficient system; LAMP, loop-mediated isotherm amplification; HUDSON, heating unextracted diagnostic samples to obliterate nucleases; DETECTR, deoxyribonucleic acid endonuclease targeted CRISPR trans reporter; SNP, single nucleotide polymorphism; SARS-CoV-2, severe acute respiratory syndrome corona virus 2; FIND-IT, fast integrated nuclease detection in tandem; SHINE, streamlined highlighting of infections to navigate epidemics.

Figure 3.

Exploitation of collateral RNA cleavage activity of Cas13a in CRISPR-based diagnostic approaches. Schematic representation of Cas13a-mediated detection of any target RNA or DNA molecule. The clinical sample is processed using methods, such as HUDSON or chemical and heat treatment, followed by extraction of nucleic acids. Target nucleic acids are pre-amplified using isothermal amplification RPA or LAMP. RNA targets are first reverse transcribed and amplified with RPA or LAMP. DNA targets are amplified directly. After amplification, the amplified targets must be transcribed using T7 RNA polymerase in case of Cas13-mediated detection. After amplification, detection is performed by adding CRISPR RNA, target and appropriate Cas enzymes. The signal is detected using either visual indicators in lateral flow strips or fluorescence monitoring in reaction tubes. Fluorescence signals can also be read with a fluorimeter for quantification. Numerous methods have been developed using clinical samples in which reaction reagents are simultaneously used in target amplification and detection. This figure was generated using BioRender (www.biorender.com). CRISPR, clustered regularly interspaced short palindromic repeats; Cas, CRISPR-associated; LAMP, loop-mediated isotherm amplification; HUDSON, heating unextracted diagnostic samples to obliterate nucleases; FAM, fluorescein amidites; NP, nanoparticle; RT, reverse transcription; RPA, recombinase polymerase amplification, -ve, negative; +ve, positive.

Figure 4.

The discovery and characterization of tandem acting and orthogonal Cas enzymes have enhanced the multiplexing of CRISPR-based diagnostic approaches. (A) Orthogonal Cas enzymes can be used simultaneously, where target recognition by one enzyme activates its collateral activity, which produces byproducts that act as an activator for the second Cas enzyme. Activation produces a stable and robust signal that can be easily detected without a pre-amplification reaction. For example, LbuCas13a and TtCsm6 can be used to detect any RNA target. The collateral activity of LbuCas13a produces 2′-3′ cyclic phosphates or linear oligonucleotides that act as an activator for TtCsm6. Stable activation of TtCsm6 produces a strong signal easily detected with a fluorimeter. (B) Orthologous Cas enzymes can be used together for multiplex detection of pathogen RNA and DNA, saving time and cost. Different Cas orthologs demonstrate different collateral activity on oligonucleotides, including dinucleotides and hexanucleotides. The figure presents four orthologous Cas enzymes used together in the presence of defined di- or oligonucleotide motifs that separate the quencher molecule. Recognition of these targets by the enzymes activates the collateral activity, which results in the generation of fluorescent signals that can be detected with a fluorimeter. This figure was generated using BioRender (www.biorender.com). Cas, clustered regularly interspaced short palindromic repeats-associated; crRNA, CRISPR RNA; ss, single-stranded; LbuCas13a, Leptotrichia buccalis Cas13a; TtCsm6, Csm6 from Thermus thermophilus; PsmCas13b, Prevotella sp. MA2016 Cas13b; CcaCas13b, Capnocytophaga canimorsus Cas13b; LwaCas13a, Leptotrichia wadei Cas13a; AsCas12a, Acidaminococcus sp. Cas12a.

Figure 5.

High throughput clustered regularly interspaced short palindromic repeats-based multiplex detection of targets. The combinatorial array reactions for the multiplex evaluation of nucleic acids uses a unique color code assigned to each PCR amplified target/sample or detection mix containing Cas13. The detection mixture comprises Cas13a, crRNA (sequence-specific) and fluorophore-quencher labeled reporter. Fluorous oil is used to emulsify color-coded solutions, which creates nl volume droplets. Samples and detection droplets are pooled together. Pooled droplets are then loaded into a microwell array chip in one step, creating all possible pairwise combinations. Fluorescence microscopy is used to identify droplet pairs in each well before they fuse using an electric field. The Cas13-based detection reaction is monitored using fluorescence microscopy. This figure was generated using BioRender (www.biorender.com). Cas, clustered regularly interspaced short palindromic repeats-associated; crRNA, CRISPR RNA.

Figure 6.

Inhibition of viral life cycle phases by Cas13. Cas13 inhibits viral replication by targeting the viral genome or genomic intermediates. Cas13 specifically targets ssRNA genomes or ssRNA intermediates produced during the life cycle of RNA or DNA viruses, including viral mRNAs. Targeting occurs within the cell once the nucleic acid is accessible to Cas13-crRNA complexes, resulting in inhibited virus release. This figure was generated using BioRender (www.biorender.com). Cas, clustered regularly interspaced short palindromic repeats-associated; ds, double-stranded; ss, singled-stranded; crRNA, CRISPR RNA.