An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing

Nat Commun. 2021 Mar 19;12(1):1739. doi: 10.1038/s41467-021-21996-6.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • COVID-19 / diagnosis*
  • COVID-19 / virology
  • COVID-19 Testing / methods*
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Cas Systems
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Endodeoxyribonucleases / genetics
  • Humans
  • Molecular Diagnostic Techniques / methods
  • Mutation
  • Nasopharynx / virology
  • Nucleic Acid Amplification Techniques / methods
  • RNA, Guide, CRISPR-Cas Systems*
  • RNA, Viral / genetics
  • SARS-CoV-2 / genetics*
  • Sensitivity and Specificity

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • RNA, Guide, CRISPR-Cas Systems
  • RNA, Viral
  • Cas12a protein
  • Endodeoxyribonucleases

Supplementary concepts

  • LAMP assay