tetano
Editor, Senior Moderator
Proc Natl Acad Sci U S A
. 2022 Jul 12;119(28):e2118260119.
doi: 10.1073/pnas.2118260119. Epub 2022 Jun 28.
Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2
Ahmed Mahas[SUP] 1 [/SUP], Tin Marsic[SUP] 1 [/SUP], Mauricio Lopez-Portillo Masson[SUP] 1 [/SUP], Qiaochu Wang[SUP] 1 [/SUP], Rashid Aman[SUP] 1 [/SUP], Cheng Zheng[SUP] 2 [/SUP], Zahir Ali[SUP] 1 [/SUP], Madain Alsanea[SUP] 3 [/SUP], Ahmed Al-Qahtani[SUP] 3 [/SUP], Bernard Ghanem[SUP] 2 [/SUP], Fatimah Alhamlan[SUP] 3 [/SUP], Magdy Mahfouz[SUP] 1 [/SUP]
Affiliations
Abstract
Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/μL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.
Keywords: CRISPR Cas13; CRISPR diagnostics; diagnostics; thermostable Cas13; transcriptome editing.
. 2022 Jul 12;119(28):e2118260119.
doi: 10.1073/pnas.2118260119. Epub 2022 Jun 28.
Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2
Ahmed Mahas[SUP] 1 [/SUP], Tin Marsic[SUP] 1 [/SUP], Mauricio Lopez-Portillo Masson[SUP] 1 [/SUP], Qiaochu Wang[SUP] 1 [/SUP], Rashid Aman[SUP] 1 [/SUP], Cheng Zheng[SUP] 2 [/SUP], Zahir Ali[SUP] 1 [/SUP], Madain Alsanea[SUP] 3 [/SUP], Ahmed Al-Qahtani[SUP] 3 [/SUP], Bernard Ghanem[SUP] 2 [/SUP], Fatimah Alhamlan[SUP] 3 [/SUP], Magdy Mahfouz[SUP] 1 [/SUP]
Affiliations
- PMID: 35763567
- DOI: 10.1073/pnas.2118260119
Abstract
Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/μL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.
Keywords: CRISPR Cas13; CRISPR diagnostics; diagnostics; thermostable Cas13; transcriptome editing.