Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively. As the COVID-19 pandemic continues, variants of the virus are emerging. Here the authors present a diagnostic assay that can detect wildtype and known variants using engineered Cas12a.
【저자키워드】 SARS-CoV-2, Biochemical assays, CRISPR-Cas9 genome editing, 【초록키워드】 Mutation, COVID-19 pandemic, variant, Infection, diagnostic, virus, variants, Predictive value, CRISPR, amplification, clinical samples, sensitivity, specificity, Viral, Positive predictive value, COVID-19 infection, Internal control, Loop-mediated isothermal amplification, nasopharyngeal, LAMP, temperature, viral genome, reaction, enzyme, healthy individuals, microliter, specimen, purified RNA, wildtype, RNA purification, transmission of SARS-CoV-2, robust, clinical sample, detect, increase, mutated, cause, exhibit, Extensive, patients with COVID-19, 【제목키워드】 variant, COVID-19 testing, robust, DNA-RNA hybrid,