Discovery of a novel enzyme that can be used in the detection of SARS-CoV-2

What is it about?

A group of researchers led by professor Magdy Mahfouz from Laboratory for Genome Engineering and Synthetic Biology at King Abdullah University of Science and Technology (KAUST) has found and characterized a thermostable member of Cas13a family of RNA-guided RNases that are extensively used in the bourgeoning field of CRISPR diagnostics. Employing numerous biochemical assays, the team determined the properties of TccCas13a enzyme from thermophilic bacterium Thermoclostridium caenicola. TccCas13a, like other members of Cas13 family, is activated in the presence of a specific RNA sequence. This activation leads to secondary activity on non-specific RNA sequences. This property can be harnessed to cleave short RNA reporters that produce signal only in the presence of the pathogen of interest. Based on their findings, the group developed a fluorescence based, POC-compatible reaction chemistry for the detection of SARS-CoV-2 named OPTIMA-dx. The new platform was coupled with a quick extraction protocol and validated on a hundred clinical samples in collaboration with Dr. Fatimah Alhamlan at King Faisal Specialist Hospital and Research Center. To facilitate data interpretation and sharing, a user-friendly mobile application was developed by a team led by Professor Bernard Ghanem from Image and Video Understanding Laboratory at King Abdullah University of Science and Technology.

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Photo by Louis Reed on Unsplash

Photo by Louis Reed on Unsplash

Why is it important?

CRISPR-Cas13 systems have so far been repurposed for various applications such as gene knock-down, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologues holds the potential of significantly expanding biotechnological applications. Notably, CRISPR-based detection methods still rely on isothermal amplification techniques such as loop-mediated isothermal amplification (LAMP) to achieve clinically relevant levels of sensitivity. However, LAMP amplification proceeds at 55-65°C, temperature that is too high for all so far characterized Cas13 enzymes. This problem was addressed in previous works by splitting the reaction in two steps. However, this led to reduced practicality and frequent false positive results due to carry-over contamination. Thermostable Cas13 orthologue from Thermoclostridium caenicola (TccCas13a) exhibits robust catalytic activities over a broad range of temperatures 37–70°C which makes it particularly suitable for solving this issue. Moreover, TccCas13a opens a path for multiplexing applications with multiple Cas enzymes. The group demonstrated this application by establishing OPTIMA-dx assay capable of detecting two targets by using TccCas13a and a related member of Cas12 family (AapCas12b). Another attractive feature of CRISPR-Cas systems is their versatility – the ability to be reprogrammed to detect virtually any pathogen or mutation of interest. Therefore, OPTIMA-dx opens prospects for the next generation of multiplexed, one-pot, point-of-care assays that can be employed for detection of any pathogen of interest.

Perspectives