Coronavirus (4) RNA tests using RT-PCR vs using CRISPR

As described in the previous article, detection of SARS-CoV-2 genetic material can be done by both RT-PCR and CRISPR. Although the detection system using CRISPR to detect the SARS-CoV-2 is not yet available in the market, a comparison between the two CRISPR platforms (DETECTR from Mammoth Biosciences and SHERLOCK from Sherlock Biosciences) and the RT-PCR (represented by a protocol developed by CDC from the US), made by the Mammoth Biosciences, let us know more about the advantages of using CRISPR in virus detection.¹

Basically, when comparing the CRISPR platform and RT-PCR, CRISPR has the advantages of shorter running time, no expensive/ special equipment needed, no highly skilled technical staff needed, and thus lower costs in general to run the test. However, the sensitivity of the CRISPR platform is not as good as that of RT-PCR. The lowest limit for detection by CRISPR is in the range of 10 to 70 copies/ul of virus in the sample, while only 3.6 to 10 copies/ul of virus in the sample is already enough to be detected by RT-PCR.

Neither of the CRISPR platform for virus RNA detection have yet been approved by the FDA in the US or by any other country, however I believe that this detection system will become more widely used than the RT-PCR in the near future. The CRISPR detection system would be particularly useful in countries whose resources are very limited, which lack instruments and technical staff to run RT-PCR.

However, one thing that we do need to be aware of is the possibility of the off-target effect generated from the two exonucleases, Cas12a and Cas13a, used in the CRISPR detection system. No study on this has been published so far. The off-target effect of Cas9 in the gene editing system generates unexpected functions of a gene and may result in genomic instability.^2,3 The off-target effect generated from the activity of Cas12a/Cas13a in the CRISPR detection system can result in an incorrect recognition of the nucleotide target, and generates a false positive result. A thorough study of the incident rate of the off-target effect in Cas12a/Cas13a system, and finding out a mechanism to decrease the off-target effect, would give us more assurance of the usability of CRISPR in genetic material detection.

References

1. “A protocol for rapid detection of the 2019 novel coronavirus SARS-CoV-2 using CRISPR diagnostics: SARS-CoV-2 DETECTR” 2nd March, 2020. https://mammoth.bio/wp-content/uploads/2020/03/Mammoth-Biosciences-A-protocol-for-rapid-detection-of-SARS-CoV-2-using-CRISPR-diagnostics-DETECTR.pdf

2. Yanfang Fu, Jennifer A Foden, Cyd Khayter, et al. “High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells” Nat Biotechnol. 2013 Sep; 31(9):822-826.

3. Seung Woo Cho, Sojung Kim, Yongsub Kim, et al. “Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases” Genome Res. 2014 Jan; 24(1): 132–141.