tetano
Editor, Senior Moderator
Microbiol Spectr
. 2024 Feb 6:e0364523.
doi: 10.1128/spectrum.03645-23. Online ahead of print. A photocontrolled one-pot isothermal amplification and CRISPR-Cas12a assay for rapid detection of SARS-CoV-2 Omicron variants
Qian Sun[SUP] #[/SUP][SUP] 1 [/SUP], Hongqing Lin[SUP] #[/SUP][SUP] 1 [/SUP], Yuan Li[SUP] 1 [/SUP], Liping Yuan[SUP] 1 [/SUP], Baisheng Li[SUP] 2 [/SUP], Yunan Ma[SUP] 1 [/SUP], Haiying Wang[SUP] 1 [/SUP], Xiaoling Deng[SUP] 2 [/SUP], Hongliang Chen[SUP] 3 [/SUP], Shixing Tang[SUP] 1 4 [/SUP]
Affiliations
CRISPR-Cas technology has widely been applied to detect single-nucleotide mutation and is considered as the next generation of molecular diagnostics. We previously reported the combination of nucleic acid amplification (NAA) and CRISPR-Cas12a system to distinguish major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. However, the mixture of NAA and CRISPR-Cas12a reagents in one tube could interfere with the efficiency of NAA and CRISPR-Cas12a cleavage, which in turn affects the detection sensitivity. In the current study, we employed a novel photoactivated CRISPR-Cas12a strategy integrated with recombinase polymerase amplification (RPA) to develop one-pot RPA/CRISPR-Cas12a genotyping assay for detecting SARS-CoV-2 Omicron sub-lineages. The new system overcomes the potential inhibition of RPA due to early CRISPR-Cas12a activation and cleavage of the target template in traditional one-pot assay using photocleavable p-RNA, a complementary single-stranded RNA to specifically bind crRNA and precisely block Cas12a activation. The detection can be finished in one tube at 39℃ within 1 h and exhibits a low limit of detection of 30 copies per reaction. Our results demonstrated that the photocontrolled one-pot RPA/CRISPR-Cas12a assay could effectively identify three signature mutations in the spike gene of SARS-CoV-2 Omicron variant, namely, R346T, F486V, and 49X, and distinguish Omicron BA.1, BA.5.2, and BF.7 sub-lineages. Furthermore, the assay achieved a sensitivity of 97.3% and a specificity of 100.0% and showed a concordance of 98.3% with Sanger sequencing results.IMPORTANCEWe successfully developed one-pot recombinase polymerase amplification/CRISPR-Cas12a genotyping assay by adapting photocontrolled CRISPR-Cas technology to optimize the conditions of nucleic acid amplification and CRISPR-Cas12a-mediated detection. This innovative approach was able to quickly distinguish severe acute respiratory syndrome coronavirus 2 Omicron variants and can be readily modified for detecting any nucleic acid mutations. The assay system demonstrates excellent clinical performance, including rapid detection, user-friendly operations, and minimized risk of contamination, which highlights its promising potential as a point-of-care testing for wide applications in resource-limiting settings.
Keywords: CRISPR-Cas12a; Omicron variant; SARS-CoV-2; genotyping; isothermal amplification; one-pot assay; photocleavage.
. 2024 Feb 6:e0364523.
doi: 10.1128/spectrum.03645-23. Online ahead of print. A photocontrolled one-pot isothermal amplification and CRISPR-Cas12a assay for rapid detection of SARS-CoV-2 Omicron variants
Qian Sun[SUP] #[/SUP][SUP] 1 [/SUP], Hongqing Lin[SUP] #[/SUP][SUP] 1 [/SUP], Yuan Li[SUP] 1 [/SUP], Liping Yuan[SUP] 1 [/SUP], Baisheng Li[SUP] 2 [/SUP], Yunan Ma[SUP] 1 [/SUP], Haiying Wang[SUP] 1 [/SUP], Xiaoling Deng[SUP] 2 [/SUP], Hongliang Chen[SUP] 3 [/SUP], Shixing Tang[SUP] 1 4 [/SUP]
Affiliations
- PMID: 38319081
- DOI: 10.1128/spectrum.03645-23
CRISPR-Cas technology has widely been applied to detect single-nucleotide mutation and is considered as the next generation of molecular diagnostics. We previously reported the combination of nucleic acid amplification (NAA) and CRISPR-Cas12a system to distinguish major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. However, the mixture of NAA and CRISPR-Cas12a reagents in one tube could interfere with the efficiency of NAA and CRISPR-Cas12a cleavage, which in turn affects the detection sensitivity. In the current study, we employed a novel photoactivated CRISPR-Cas12a strategy integrated with recombinase polymerase amplification (RPA) to develop one-pot RPA/CRISPR-Cas12a genotyping assay for detecting SARS-CoV-2 Omicron sub-lineages. The new system overcomes the potential inhibition of RPA due to early CRISPR-Cas12a activation and cleavage of the target template in traditional one-pot assay using photocleavable p-RNA, a complementary single-stranded RNA to specifically bind crRNA and precisely block Cas12a activation. The detection can be finished in one tube at 39℃ within 1 h and exhibits a low limit of detection of 30 copies per reaction. Our results demonstrated that the photocontrolled one-pot RPA/CRISPR-Cas12a assay could effectively identify three signature mutations in the spike gene of SARS-CoV-2 Omicron variant, namely, R346T, F486V, and 49X, and distinguish Omicron BA.1, BA.5.2, and BF.7 sub-lineages. Furthermore, the assay achieved a sensitivity of 97.3% and a specificity of 100.0% and showed a concordance of 98.3% with Sanger sequencing results.IMPORTANCEWe successfully developed one-pot recombinase polymerase amplification/CRISPR-Cas12a genotyping assay by adapting photocontrolled CRISPR-Cas technology to optimize the conditions of nucleic acid amplification and CRISPR-Cas12a-mediated detection. This innovative approach was able to quickly distinguish severe acute respiratory syndrome coronavirus 2 Omicron variants and can be readily modified for detecting any nucleic acid mutations. The assay system demonstrates excellent clinical performance, including rapid detection, user-friendly operations, and minimized risk of contamination, which highlights its promising potential as a point-of-care testing for wide applications in resource-limiting settings.
Keywords: CRISPR-Cas12a; Omicron variant; SARS-CoV-2; genotyping; isothermal amplification; one-pot assay; photocleavage.