CASCADE: Naked eye-detection of SARS-CoV-2 using Cas13a and gold nanoparticles

The COVID-19 pandemic has brought to light the need for fast and sensitive detection methods to prevent the spread of pathogens. The scientific community is making a great effort to design new molecular detection methods suitable for fast point-of-care applications. In this regard, a variety of approaches have been developed or optimized, including isothermal amplification of viral nucleic acids, CRISPR-mediated target recognition, and read-out systems based on nanomaterials. Herein, we present CASCADE (CRISPR/CAS-based Colorimetric nucleic Acid DEtection), a sensing system for fast and specific naked-eye detection of SARS-CoV-2 RNA. In this approach, viral RNA is recognized by the LwaCas13a CRISPR protein, which activates its collateral RNase activity. Upon target recognition, Cas13a cleaves ssRNA oligonucleotides conjugated to gold nanoparticles (AuNPs), thus inducing their colloidal aggregation, which can be easily visualized. After an exhaustive optimization of functionalized AuNPs, CASCADE can detect picomolar concentrations of SARS-CoV-2 RNA. This sensitivity is further increased to low femtomolar (3 fM) and even attomolar (40 aM) ranges when CASCADE is coupled to RPA or NASBA isothermal nucleic acid amplification, respectively. We finally demonstrate that CASCADE succeeds in detecting SARS-CoV-2 in clinical samples from nasopharyngeal swabs. In conclusion, CASCADE is a fast and versatile RNA biosensor that can be coupled to different isothermal nucleic acid amplification methods for naked-eye diagnosis of infectious diseases. Graphical Image 1

CRISPR/Cas technology as a promising weapon to combat viral infections.

The versatile clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has emerged as a promising technology for therapy and molecular diagnosis. It is especially suited for overcoming viral infections outbreaks, since their effective control relies on an efficient treatment, but also on a fast diagnosis to prevent disease dissemination. The CRISPR toolbox offers DNA- and RNA-targeting nucleases that constitute dual weapons against viruses. They allow both the manipulation of viral and host genomes for therapeutic purposes and the detection of viral nucleic acids in "Point of Care" sensor devices. Here, we thoroughly review recent advances in the use of the CRISPR/Cas system for the treatment and diagnosis of viral deleterious infections such as HIV or SARS-CoV-2, examining their strengths and limitations. We describe the main points to consider when designing CRISPR antiviral strategies and the scientific efforts to develop more sensitive CRISPR-based viral detectors. Finally, we discuss future prospects to improve both applications. Also see the video abstract here: https://www.youtube.com/watch?v=C0z1dLpJWl4.