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Chinese Researchers Develop CRISPR-Based Isothermal COVID-19 Diagnostic

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NEW YORK – Researchers in China have developed a new, CRISPR-based coronavirus test that could further fill the need for fast, inexpensive, and convenient viral detection testing.

In a study published on Thursday in PLOS Pathogens, the scientists described the assay, which combines isothermal amplification and CRISPR-Cas13a to detect SARS-CoV-2 with near single-copy sensitivity.

The researchers also compared their method — which they named CRISPR-COVID — to metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR)-based tests, noting that it is faster and requires less complex instrumentation. According to co-corresponding author Teng Xu, founder and chief technology officer of Vision Medicals Center for Infectious Diseases in Guangdong, this study is also the first to assess a CRISPR-based COVID assay in China.

Xu and his collaborators from the Chinese Academy of Medical Sciences & Peking Union Medical College, the Guangdong Academy of Medical Sciences Guangzhou, and elsewhere noted that their CRISPR-COVID assay doesn't require a thermocycler to run. That cuts the turnaround time to about 40 minutes and would also make this test a good option for low-resource settings.

"These features would make it not only beneficial for on-the-spot testing, but also get around the PCR instrument bottleneck in lab settings when dealing with high-volume testing," Xu said.

In order to develop their assay, the researchers obtained total RNA samples from 61 patients suspected of having COVID-19 and subjected them to mNGS analysis. They were able to confirm 52 cases with read numbers mapping to SARS-CoV-2 across six orders of magnitude, indicating a high degree of variation in the patients' viral loads.

Using this data, the researchers aimed to identify target regions of SARS-CoV-2 by searching for sequences that were within the Orf1ab, N, or E genes of the viral genome; conserved among strains of the novel virus; and differentiable from other pathogenic coronaviruses. By analyzing the genetic similarity among the SARS-CoV-2 genomes and other pathogenic coronaviruses, they identified two potential target sequences in Orf1ab and one in the N gene.

Once they had the data in hand, they believed that the best technological platform for a rapid, highly sensitive, and simple-to-use assay was a combination of the recombinase polymerase amplification (RPA) isothermal DNA amplification technique and CRISPR-Cas-mediated enzymatic signal amplification for improved sensitivity. The isothermal nature of the assay obviated the need for complex instruments.

Based on the three potential target sequences they had already identified, the researchers designed and screened multiple sets of RPA primers and CRISPR guide RNAs. Among these, the set that targeted Orf1ab showed the best overall performance for sensitivity and specificity. The researchers then sought to determine CRISPR-COVID's analytic sensitivity at various concentrations and found that it consistently detected 7.5 copies per reaction of SARS-CoV-2 in all 10 replicates, 2.5 copies per reaction in six out of 10, and 1.25 copies per reaction in two out of 10 runs, indicating a near single-copy sensitivity.

To confirm the specificity, they tested CRISPR-COVID with DNA from human cells, as well as a panel of microbes including bacteria commonly found in respiratory infections such as S. pneumonia, H. influenza, M. pneumonia, C. pneumonia, and B. pertusiss; human coronaviruses such as H-Co-V-OC43, HCoV-NL63, HCoV-HKU1, and HCoV-229E; other viruses commonly found in respiratory infections such as Adenovirus Type-3, H. Influenza B, H. influenza A, HPIV-1, and RSV-A; and other bacteria such as S. mitis, S. pyogenes, S. aureus, E. coli, and E. facecaiis. None of them triggered a false positive reaction.

Once their analytical assessment was complete, the researchers looked to further evaluate the diagnostic potential of CRISPR-COVID in clinical specimens by testing it on 114 RNA samples, which consisted of the previously sequenced 61 cases of suspected COVID-19, 17 SARS-CoV-2/hCoV-positive cases, and 36 samples from healthy subjects.

CRISPR-COVID demonstrated a sensitivity of 100 percent by detecting all 52 COVID-19 cases. It didn't signal any false positives among the 62 negative cases, including the hCoV-infected samples, suggesting a promising clinical sensitivity and specificity, the researchers said.

When they compared the diagnostic performance of CRISPR-COVID to mNGS and RT-PCR, they found that their test had a specificity of 100 percent. PCR testing was able to detect the virus in 90.4 percent of the positive cases. They also noted that the five false negative results signaled by the PCR test had a much lower titer of the virus than the other samples. In comparison, CRISPR showed a greater sensitivity by detecting all 52 COVID-19 cases.

Further, the researchers said, CRISPR-COVID's turnaround time of 40 minutes was the lowest among the three methods. It included 30 minutes of DNA amplification and 10 minutes of Cas reaction. PCR required about an hour and a half for a complete run, and mNGS took about 20 hours to complete.

Xu noted that the study was done using several sample types — nasal swabs, sputum, and bronchoalveolar lavage fluid samples. "Analytically speaking," he added, "[CRISPR-COVID] would work with saliva samples, but more clinical evaluation is needed. We have been expanding our testing on both positive and negative samples."

Importantly, CRISPR-based testing is also a financially competitive alternative to PCR testing. "At the current research scale, the material costs of a CRISPR-COVID test run at [about] $3.50" per test," Xu said. "We envisage the production cost after scaling up would run close to, but likely slightly higher, than PCR." In their study, the researchers specified that the costs would likely drop significantly at scaled-up production to about $.70 per test.

For now, Xu said, the researchers haven't decided on a strategy for regulatory approval, given that COVID-19 has been "well controlled" in China. But they believe the technology has potential for the long term, and given its demonstrated ability to differentiate specific viral signals from each other, Xu said, "[we] plan on integrating the assay into an automated, sample-in-result-out system, which encompasses other common respiratory pathogens." 

In the US, several companies have also started to forge ahead with CRISPR-based tests for the virus. The assays have different capabilities that make them suitable for a range of settings.

Sherlock Biosciences, for example, partnered with Binx Health in July to develop a CRISPR-based rapid, point-of-care diagnostic test for COVID-19. The companies plan to combine the Binx Io diagnostic platform with SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) CRISPR technology to create a test that's designed to provide rapid and accurate results in a single patient visit across many diverse CLIA-waived settings. In May, Sherlock Bio was also the first company to receive Emergency Use Authorization from the US Food and Drug Administration for its Sherlock CRISPR SARS-CoV-2 kit, which is designed for use in high-volume CLIA laboratories and hospitals.

Meanwhile, Mammoth Biosciences is also developing CRISPR-based COVID tests for the POC and at-home settings, in partnership with the consumer healthcare arm of pharmaceutical company GlaxoSmithKline. In April, the company said it had developed a diagnostic assay for SARS-CoV-2 based on its DETECTR platform that could be run in 30 to 45 minutes with the same sensitivity and specificity as qRT-PCR tests. The test received EUA from the FDA in July.

Also in July, a startup called Caspr Biotech that's based in Argentina and the US said it had developed a COVID diagnostic that combined CRISPR and a lyophilized format of isothermal amplification. The idea behind the test, according to the company, was that it allowed for accurate detection of the virus with minimal external equipment, making it ideal for countries with limited resources, much like CRISPR-COVID.