CRISPR-Cas, Argonaute proteins and the emerging landscape of amplification-free diagnostics

Abstract

Polymerase Chain Reaction (PCR) is the reigning gold standard for molecular diagnostics. However, the SARS-CoV-2 pandemic reveals an urgent need for new diagnostics that provide users with immediate results without complex procedures or sophisticated equipment. These new demands have stimulated a tsunami of innovations that improve turnaround times without compromising the specificity and sensitivity that has established PCR as the paragon of diagnostics. Here we briefly introduce the origins of PCR and isothermal amplification, before turning to the emergence of CRISPR-Cas and Argonaute proteins, which are being coupled to fluorimeters, spectrometers, microfluidic devices, field-effect transistors, and amperometric biosensors, for a new generation of nucleic acid-based diagnostics.

Keywords: Argonaute; CRISPR-Cas; Molecular diagnostics; Nucleic acid detection; TnpB/IscB.

Graphical abstract

Fig. 1

Evolution of molecular diagnostics. A decade of technological and methodological improvements to PCR resulted in a sensitive and specific amplification method that is the gold-standard of molecular diagnostics. However, guidelines that facilitated reproducible and reliably interpretable qPCR assays were established over a 16-year period. Isothermal amplification methods developed in the 90 s and early 2000 s eliminate the need for thermocyclers and reduce reaction times. While CRISPR-dx and pAgo-dx were initially used as secondary methods to confirm results from isothermal amplification, current research aims to combine CRISPR-dx or pAgo-dx, with novel detection methodologies, signaling cascades and protein circuits that do not rely on pre-amplification.

Fig. 2

Sensitive nucleic acid detection using programmable target recognition and activated collateral nucleases. (A) RNA-guided target recognition by Cas13 (top) or Cas12 (bottom) allosterically activates a collateral nuclease activity, which cleaves a reporter RNA or DNA. Reporter nucleic acids can be labeled with different functional groups that result in a variety of readouts including fluorescent, colorimetric, electrical and Raman scattering. (B) Dilution of the diagnostic reaction into microchambers leads to a digital count (0 or 1) of target-containing chambers, enabling the absolute quantification of targets in the original sample and improving sensitivity 10,000-fold over bulk reactions. (C) Amperometric sensors detect the reduction or oxidation of a gold electrode, which results in a change in current. To detect collateral nuclease activity on an amperometric sensor, a redox-active molecule (purple sphere) is tethered to a gold electrode by a nucleic acid tether. Cleavage of the nucleic acid tether changes the current across an electrode. (D) Surface-enhanced Raman scattering (SERS) improves the sensitivity of lateral flow assays > 1,000-fold over visual inspection alone. (E) Feedback amplification circuits of CRISPR enzymes can be designed to mimic the exponential “chain reaction” of DNA amplification that occurs during PCR, leading to a 1,000,000-fold increase in sensitivity.

Fig. 3

CRISPR-mediated signaling inspires new CRISPR-dx applications. (A) Type III (i.e., Cmr and Csm) CRISPR complexes are used for the direct detection of RNA. Target RNA binding allosterically activates a Cas10 polymerase subunit (Cas10 pol) in Csm or Cmr that produces a mixture of cyclic oligoadenylate molecules (cA₂ – cA₆). Cyclic oligoadenylate molecules bind and activate CRISPR-associated effectors including RNases, DNases, and peptidases, which can be repurposed to cleave RNA, DNA, peptides, or proenzyme reporters. (B) The collateral nuclease activity of Cas13 produces 2′3′-cyclic phosphates at the 3′ ends of cleaved RNAs. Cas13-mediated cleavage of a specially designed reporter RNA thereby produces an artificial linear ligand that activates CRISPR-associated effectors, resulting in > 100-fold increase in sensitivity. (C) Cas13-mediated nicking of an inhibiting RNA loop makes an RNA-DNA hybrid duplex accessible for Cas12 RNA-guided DNA binding. One Cas13 thereby activates many Cas12 complexes, which in turn cleave many fluorescent reporter DNAs, increasing the sensitivity 1000-fold over direct Cas13-based detection.

Fig. 4

Target-guided nucleases and sensitive methods to detect target binding. (A) Cas9 can be repurposed to detect RNA in a multiplexed protocol, by using reprogrammed tracrRNAs (Rptrs) that anneal to the target RNA and coerce the adjacent RNA sequence to behave as a typical RNA guide that results in cleavage of a complementary DNA reporter. The presence of many different RNA targets is determined using gel electrophoresis or a Bioanalyzer. Future microarray-based detection methods may enable multiplexed detection of millions of RNAs. (B) DNA-guided prokaryotic Argonautes cleave dsDNA targets into fragments that are recognized as new gDNAs (guide DNAs), which direct pAgos to cleave complementary DNA reporters. Unlike target-guided Cas9, target-guided Argonautes are multi-turnover enzymes that cleave many reporter DNAs per target-derived guide. (C) RNA-guided Cas9 can be repurposed to detect complementary dsDNA target. Cas9-bound dsDNA induces a large change in the electrical properties of the tethered graphene channel, resulting in rapid and sensitive electrical detection of DNA. (D) Two RNA-guided Cas9s, each fused to different halves of a split reporter enzyme can be repurposed to detect dsDNA. Stable association of each Cas9 to the adjacent complementary sites results in the reconstitution of the reporter enzyme (e.g., Luciferase).

Fig. 5

The emerging landscape of pre-amplification-independent CRISPR-dx and pAgo-dx. Unique and sensitive Cas12-, Cas13-, Type III CRISPR-, Cas9- and pAgo-based diagnostic methodologies that do not rely on preamplification compared to the CDC’s recommended RT-qPCR protocol for SARS-CoV-2 , , , , , , , , , , , . Data and citations included in Table S2.