Cas13 engineered to simplify detection of new crowns and other diseases

A new CRISPR-based engineered method accurately finds RNA for the COVID-19 virus SARS-CoV-2. This highly sensitive detector promises to make detection of COVID-19 and other diseases fast and easy.

Cas13 engineered to simplify detection of new crowns and other diseases

To improve its ability to detect trace amounts of SARS-CoV-2 virus in biological samples, collaborators from Rice University and the University of Connecticut further designed the RNA-editing CRISPR-Cas13 system. A huge advantage of the system is that it reportedly does not require the time-consuming RNA extraction and amplification steps necessary in gold-standard PCR tests.

This new platform was very successful compared to the PCR test. In fact, in tests on clinical samples directly from nasal swabs, it found 10 out of 11 positives and no false positives. The scientists showed that their technique could detect attomolar (10-18) concentration of SARS-CoV-2 signs.

Cas13 engineered to simplify detection of new crowns and other diseases

Cas13, like its better known cousin Cas9, is part of the phage system that is the natural defense of bacteria against invasion. Since its discovery, CRISPR-Cas9 has been used by scientists to edit living DNA genomes and has shown great promise for treating and even curing diseases.

In addition, it can be used in other ways. cas13 itself can be enhanced with guide RNA and thus used to find and clip target RNA sequences, while also looking for "appendages", in this case, the presence of viruses like SARS-CoV-2.

"The engineered Cas13 protein in this work can be easily adapted to other previously established platforms," said researcher Xue Sherry Gao, "and the stability and robustness of the engineered Cas13 variants make them more suitable for low-resource PCR machines in low-cost environment areas for point-of-care diagnostics."

Another researcher, Jie Yang, said the wild-type Cas13, from a bacterium Leptotrichia wadei, was unable to detect attomolar levels of viral RNA in the 30- to 60-minute time frame, but the enhanced version created at Rice did the job in about half an hour and detected SARS-CoV-2 at much lower concentrations than previous tests.

She went on to point out that the key is a well-hidden, flexible hairpin loop near the Cas13 active site. "It is in the middle of the protein, near the catalytic site, and determines the activity of Cas13. Because Cas13 is large and dynamic, it is challenging to find a site that inserts into another functional domain."

Cas13 engineered to simplify detection of new crowns and other diseases

The team fused seven different RNA-binding domains to the loop, with two of the complexes having a clear advantage. When they found their target, these proteins fluoresced and showed the presence of the virus.

"We can see that the increased activity is five or six times greater than that of wild-type Cas13. That number seems small, but it's pretty impressive in one step of protein engineering," Yang said, "but it's still not enough for detection, so we moved the whole assay from a fluorescent plate reader (which is quite large and unusable in a low-resource environment) to an electrochemical sensor that is more sensitive and can be be used for point-of-care diagnostics."

Yang said the engineered protein was five orders of magnitude (100,000-fold) more sensitive in detecting viruses compared to the wild-type protein when using an off-the-shelf sensor.

The lab hopes to apply its technology to paper strips like those used in home COVID-19 antibody tests, though this will require greater sensitivity and accuracy. "We hope this will make testing more convenient and provide lower costs for many targets," Gao said.

The researchers are also working on improved detection of Zika, dengue and Ebola viruses and predictive biomarkers for cardiovascular disease. Their work may lead to rapid diagnosis of the severity of COVID-19.

"Different viruses have different sequences. We can design guide RNA to target a specific sequence that we can then detect, and that's the power of the CRISPR-Cas13 system," Yang said.

However, since the project started just at the time of the COVID-19 pandemic, SARS-CoV-2 was a natural focus.

"As a combined effort of structural biology, protein engineering and biomedical device development, we are very excited about this work," Gao added, "and I am very grateful to my lab members and collaborators for all their efforts."