CRISPR’s claim to fame may be gene editing—and turning the scientific community on its head when it first debuted—but it may have another trick up its sleeve.
Recent studies have indicated CRISPR tools have the potential for in vitro diagnostics, something Chinese scientists have leveraged to develop a 100% accurate COVID-19 test that can be mass manufactured for 70 cents.
Currently, metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) have been the most commonly used molecular methods for diagnosing COVID-19, but each has its own limitations. For example, sequencing is costly and has a turnaround time of nearly one day, while RT-PCR requires specialized equipment and is difficult to deploy at a large scale. As the world takes precautions to get back to some sort of normalcy, there are increased demands for rapid, sensitive and inexpensive diagnostics.
“A tool kit of rapid diagnostics faster than typical RT-PCR is in great demand to circumvent the bottlenecks in assay turnaround time and reagent supply for COVID-19 testing,” the researchers write in their study published in PLOS Pathogens. “To address the expanding clinical needs, we developed CRISPR-COVID, a rapid assay for SARS-CoV-2 detection based on Cas13a.”
Through genomic sequencing, scientists Tieying Hou, Weiqi Zeng, Minling Yang and Wenjing Chen identified two regions of SARS-CoV-2 that differentiates it from other pathogens, including other coronaviruses. With two targets in hand, the team took advantage of the sensitivity of CRISPR/Cas—specifically the programmability of CRISPR/Cas13a—to create an isothermal-based assay that eliminates the need for sophisticated analytical instruments.
Analytically, CRISPR-COVID (as the researchers have named the assay), showed a near single-copy sensitivity in the study. Even testing against human cells infected with a variety of microbes as interference samples, CRISPR-COVID did not have one false positive.
For the diagnostic portion of the study, the researchers used a total of 114 RNA samples—61 suspected COVID-19 cases (52 confirmed, 9 ruled-out by mNGS), 17 SARS-CoV-2-/hCoV+ cases and 36 samples from healthy subjects. CRISPR-COVID demonstrated a sensitivity of 100% by detecting all 52 COVID-19 cases. No false positives were found in all 62 negative cases, including the hCoV-infected samples.
Comparatively, PCR tests detected the virus in 90% of positive samples, and requires 1.5 hours for a completion run. mNGS takes approximately 20 hours, which includes 8 hours of library preparation, 10 hours of sequencing and 2 hours of bioinformatic analysis. On the other hand, the turnaround time of CRISPR-COVID is 40 minutes, including 30 minutes of DNA amplification and 10 minutes of Cas reaction.
A crucial advantage of CRISPR-COVID is its ability to be used in low-resource settings that can’t currently support the thermo cycler needed for PCR-based tests. Another advantage is the price. According to the study, at research scale, the material costs of a CRISPR-COVID test is about $3.50, which the researchers say can be dramatically reduced at population scale to below 70 cents.
“The current discovery of the collateral activity of certain Cas family members, provides a great opportunity to take advantage of both the sensitivity of an isothermal assay and the specificity of the CRISPR system, suggesting CRISPR-COVID as a competitive alternative not only technologically but also financially,” conclude the researchers.
Photo credit: NIH