An editorial advocating a new method of CRISPR testing was published in NEJM by a team of scientists from MIT, University of Washington, and Brigham & Women’s Hospital.
MIT Scientists Propose CRISPR Testing for SARS-CoV-2
Correspondence published yesterday in the New England Journal of Medicine advocates for exploring a new path in SARS-CoV-2 testing: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based diagnostics.
The editorial was published by a team of scientists including investigators from Massachusetts Institute of Technology, University of Washington, and Brigham and Women’s Hospital.
“Methods such as SHERLOCK (specific high-sensitivity enzymatic reporter unlocking), which typically use a two-step process (target amplification followed by CRISPR-mediated nucleic acid detection), have been used to detect SARS-CoV-2,” authors explain.
These approaches, however, have a problem.
Methods like SHERLOCK are more complicated than those used in existing coronavirus point-of-care testing; they rely on multiple liquid-handling steps and an RNA extraction step. This increases the risk of cross-contamination in samples.
In the editorial, though, investigators describe a simple test for detection of SARS-CoV-2 with sensitivity similar to that of reverse-transcription—quantitative polymerase-chain-reaction (RT-qPCR) assays.
Labeled “STOP” (SHERLOCK testing in one pot), the investigational method is a streamlined assay which synthesizes extraction of viral RNA with “isothermal amplification and CRISPR-mediated detection.”
The test can be performed at a constant temperature with minimal equipment, and in less than an hour, according to the team.
The scientists explain that the integration of isothermal amplification with CRISPR-mediated detection requires the development of a “common reaction buffer” that would work with both steps.
To amplify viral RNA, they chose reverse transcription followed by what is called “loop-mediated isothermal amplification” (LAMP).
This was because the required LAMP reagents are commonly available and use defined buffers that are amenable to Cas enzymes.
LAMP operates at high temperatures and requires a “thermostable Cas enzyme such as Cas12b from Alicyclobacillus acidiphilus (AapCas12b)” according to the scientists.
The team systematically evaluated LAMP primer sets and corresponding guide RNAs (according to the researchers, a guide RNA helps AapCas12b recognize and cut target DNA) to identify the best combination, encoding the SARS-CoV-2 nucleocapsid protein, in a single-pot reaction mixture.
"In blinded testing at an external laboratory at the University of Washington, we tested 202 SARS-CoV-2—positive and 200 SARS-CoV-2–negative nasopharyngeal swab samples obtained from patients. These samples were prepared by adding 50 μl of swab specimens obtained from patients with Covid-19 to a clean swab, in accordance with the recommendation of the Food and Drug Administration for simulating whole swabs for regulatory applications. This testing showed that STOPCovid.v2 had a sensitivity of 93.1% and a specificity of 98.5%."
According to the team, STOPCovid.v1 detection was a success, and presents an interesting new path to consider for testing research moving forward.