CRISPR-Cas12a-Based Detection for the Major SARS-CoV-2 Variants of Concern.
Yuanhao Liang,Hongqing Lin,Lirong Zou,Jianhui Zhao,Baisheng Li,Haiying Wang,Jing Lu,Jiufeng Sun,Xingfen Yang,Xiaoling Deng,Shixing Tang +10 more
TLDR
Wang et al. as discussed by the authors designed and analytically validated a CRISPR-Cas12a system for direct detection of SARS-CoV-2 VOCs, which may be more transmissible and/or more virulent and could escape immunity obtained through infection or vaccination.Abstract:
A big challenge for the control of COVID-19 pandemic is the emergence of variants of concern (VOCs) or variants of interest (VOIs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be more transmissible and/or more virulent and could escape immunity obtained through infection or vaccination. A simple and rapid test for SARS-CoV-2 variants is an unmet need and is of great public health importance. In this study, we designed and analytically validated a CRISPR-Cas12a system for direct detection of SARS-CoV-2 VOCs. We further evaluated the combination of ordinary reverse transcription-PCR (RT-PCR) and CRISPR-Cas12a to improve the detection sensitivity and developed a universal system by introducing a protospacer adjacent motif (PAM) near the target mutation sites through PCR primer design to detect mutations without PAM. Our results indicated that the CRISPR-Cas12a assay could readily detect the signature spike protein mutations (K417N/T, L452R/Q, T478K, E484K/Q, N501Y, and D614G) to distinguish alpha, beta, gamma, delta, kappa, lambda, and epsilon variants of SARS-CoV-2. In addition, the open reading frame 8 (ORF8) mutations (T/C substitution at nt28144 and the corresponding change of amino acid L/S) could differentiate L and S lineages of SARS-CoV-2. The low limit of detection could reach 10 copies/reaction. Our assay successfully distinguished 4 SARS-CoV-2 strains of wild type and alpha (B.1.1.7), beta (B.1.351), and delta (B.1.617.2) variants. By testing 32 SARS-CoV-2-positive clinical samples infected with the wild type (n = 5) and alpha (n = 11), beta (n = 8), and delta variants (n = 8), the concordance between our assay and sequencing was 100%. The CRISPR-based approach is rapid and robust and can be adapted for screening the emerging mutations and immediately implemented in laboratories already performing nucleic acid amplification tests or in resource-limited settings. IMPORTANCE We described CRISPR-Cas12-based multiplex allele-specific assay for rapid SARS-CoV-2 variant genotyping. The new system has the potential to be quickly developed, continuously updated, and easily implemented for screening of SARS-CoV-2 variants in resource-limited settings. This approach can be adapted for emerging mutations and implemented in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests using existing resources and extracted nucleic acid.read more
References
More filters
Journal ArticleDOI
MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability
Kazutaka Katoh,Daron M. Standley +1 more
TL;DR: This version of MAFFT has several new features, including options for adding unaligned sequences into an existing alignment, adjustment of direction in nucleotide alignment, constrained alignment and parallel processing, which were implemented after the previous major update.
Journal ArticleDOI
A new coronavirus associated with human respiratory disease in China.
Fan Wu,Su Zhao,Bin Yu,Yan-Mei Chen,Wen Wang,Zhi gang Song,Yi Hu,Zhao Wu Tao,Jun Hua Tian,Yuan Yuan Pei,Ming Li Yuan,Yu Ling Zhang,Fa Hui Dai,Yi Liu,Qi Min Wang,Jiao Jiao Zheng,Lin Xu,Edward C. Holmes,Edward C. Holmes,Yong-Zhen Zhang,Yong-Zhen Zhang +20 more
TL;DR: Phylogenetic and metagenomic analyses of the complete viral genome of a new coronavirus from the family Coronaviridae reveal that the virus is closely related to a group of SARS-like coronaviruses found in bats in China.
Journal ArticleDOI
Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein.
Alexandra C. Walls,Young-Jun Park,M. Alejandra Tortorici,M. Alejandra Tortorici,Abigail Wall,Andrew T. McGuire,Andrew T. McGuire,David Veesler +7 more
TL;DR: It is demonstrating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination, and it is shown that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of Sars- coV- 2 S and SARS S bind with similar affinities to human ACE2, correlating with the efficient spread of SATS among humans.
Journal ArticleDOI
Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction
TL;DR: A new software tool called Primer-BLAST is presented to alleviate the difficulty in designing target-specific primers and combines BLAST with a global alignment algorithm to ensure a full primer-target alignment and is sensitive enough to detect targets that have a significant number of mismatches to primers.
Journal ArticleDOI
Detection of SARS-CoV-2 in Different Types of Clinical Specimens.
TL;DR: Results of PCR and viral RNA testing for SARS-CoV-2 in bronchoalveolar fluid, sputum, feces, blood, and urine specimens from patients with COVID-19 infection in China are described to identify possible means of non-respiratory transmission.