In a recent PNAS publication, a Stanford University research group presented their newly developed diagnosis method for the detection of SARS-CoV-2. By utilizing CRISPR-Cas12, the scientists created a sensitive method for the detection of viral RNA. They also streamlined and integrated multiple laborious steps, including RNA extraction from clinical samples and the reverse transcription of RNA into DNA by using microfluidics technology. An additional advantage of microfluidics is that it reduces the time for biochemical reactions. The result is a faster and still sensitive technique that could have commercial applications.
Currently, the gold standard for diagnosis of COVID-19 is a method called Reverse Transcription – Polymerase Chain Reaction (RT-PCR). It can detect as little as 1 copy of viral DNA in 1 microliter of reaction. Although very sensitive, this method is lengthy as it requires the extraction of viral RNA from patient samples, followed by the transcription of the RNA into DNA before proceeding to the RT-PCR test. Additional methods, such as antibody testing, are less reliable than RT-PCR and cannot be used for the early detection of the virus. New methods that reduce the time to results, reduce the volume of reagents necessary, and maintain a high-level sensitivity for early detection of the virus are necessary.
CRISPR as a Diagnosis Tool
CRISPR is a broad term referring to the “CRISPR-Cas” family of proteins, which are part of the immune mechanism of bacteria and archaea organisms. The most common type of CRISPR use in laboratories is CRISPR-Cas9 since it has the potential to be used for therapeutic applications.
All members of this family can cut specific nucleic acid sequences based on a guide RNA (gRNA). However, some members of the CRISPR-Cas family of proteins can become “activated” after the first specific cut and then indiscriminately cut single-stranded DNA. Scientists have adopted this “activated” stage for diagnosis proposes, particularly for the CRISPR-Cas12 system. Back in March 2020, a protocol was developed for the detection of SARS-CoV-2, which is the virus responsible for COVID19. The protocol involved using CRISPR-Cas12 to specifically cleave the viral DNA, leading to the “activation” CRISPR-Cas12. At this stage, the protein would cleave synthetic single-stranded DNA, which would release a marker to be used as a readout for target detection. However, the protocol took longer than one hour, consume large volumes of reagents, and was hard to automate.
Combining Microfluidics and CRISPR
Microfluidics is a technology that manipulates small amounts of fluids in chips, which allows performing precise reactions using a fraction of the normal volume of reagents. Taking advantage of this technology, the Stanford team was able to extract RNA from a nasal swab from COVID19 patients and convert this RNA into DNA. At the same time, this technique accelerated the CRISPR-Cas12 enzymatic activity.
In their report, the scientists were able to reduce the time from nasal swab collection to results to around 35 min. Furthermore, this technique’s detection limit was an estimate of 10 copies of viral DNA in 1 microliter of reaction while using 100 times lower volume than previous CRISPR-Cas12 assays. To validate this new assay, the scientist tried to detect the virus in 32 infected and 32 non-infected individuals, which were also diagnosed using the RT-PCR technique. The results showed the CRISPR-Cas12 assay correctly detected 30 out of 32 infected individuals with no false-positive results. Although further development is necessary to enable the use of this assay at the point of care, including in low-resource settings, this assay has the potential to reduce the time and cost of diagnosis for COVID19.
By Daniel Ojeda, Ph.D.
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