Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a

Yang, Jun; Barua, Nilakshi; Rahman, Md. Nannur; Li, Carmen; Lo, Norman; Yeong, Kai Yan; Tsang, Tsz Fung; Yang, Xiao; Cheung, Yuk-Yam; Tsang, Alan K.L.; Chan, RW; Leung, Eddie Chi Man; Chan, Paul K.S.; Ip, Margaret

doi:10.1128/spectrum.03260-22

Cited by 9 publications

(6 citation statements)

References 38 publications

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“…Several studies have developed methods to distinguish among different strains of SARS-CoV-2. Isothermal amplification techniques such as Recombinase Polymerase Amplification (RPA), 15,16 Recombinase-Aided Amplification (RAA), 17 and Loop-mediated Isothermal Amplification (LAMP) 18 are rapid and simple methods that can be used for POC detection. On the contrary, there are some limitations of isothermal amplification methods, such as the secondary structure of primers (longer than 30 bp for RPA and RAA), complicated primer design (at least three primer pairs for LAMP), optimized primer concentration, suitable incubation temperature, and downstream detection method.…”

Section: Discussionmentioning

confidence: 99%

“Nano COVID-19”: Nanopore sequencing of spike gene to identify SARS-CoV-2 variants of concern

Nimsamer,

Sawaswong,

Klomkliew

et al. 2023

Exp Biol Med (Maywood)

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Coronavirus disease 2019 (COVID-19) is a worldwide pandemic infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). World Health Organization (WHO) has defined the viral variants of concern (VOC) which cause more severe disease, higher transmissibility, and reduced vaccine efficacy. In this study, the “Nano COVID-19” workflow based on Oxford nanopore sequencing of the full-length spike gene combined with flexible data analysis options was developed to identify SARS-CoV-2 VOCs. The primers were designed to cover the full-length spike gene and can amplify all VOC strains. The results of VOC identification based on phylogenetic analysis of the full-length spike gene were comparable to the whole genome sequencing (WGS). Compared to the standard VOC identification pipeline, the fast analysis based on Read Assignment, Mapping, and Phylogenetic Analysis in Real Time (RAMPART) and the user-friendly method based on EPI2ME yielded 89.3% and 97.3% accuracy, respectively. The EPI2ME pipeline is recommended for researchers without bioinformatic skills, whereas RAMPART is more suitable for bioinformaticians. This workflow provides a cost-effective, simplified pipeline with a rapid turnaround time. Furthermore, it is portable to point-of-care SARS-CoV-2 VOC identification and compatible with large-scale analysis. Therefore, “Nano COVID-19” is an alternative viral epidemic screening and transmission tracking workflow.

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Section: Discussionmentioning

confidence: 99%

“Nano COVID-19”: Nanopore sequencing of spike gene to identify SARS-CoV-2 variants of concern

Nimsamer,

Sawaswong,

Klomkliew

et al. 2023

Exp Biol Med (Maywood)

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“…However, the detection accuracy and stability may not be as strong compared to other methods 89 . Nevertheless, pre‐amplification‐based CRISPR‐Cas13 assays require longer incubation time and additional reagents for in vitro transcription (IVT) compared to Cas12, leading to increased turnaround time and reagent cost 74 . Moreover, since the reporter of Cas13 is ssRNA, its degradation may lead to false positive results 83 …”

Section: Detection Of Vocs Using Crispr Systemmentioning

confidence: 99%

“…RRCd procedures can be performed at 37°C without the need for RNA extraction and purification. Yang et al 74 developed a rapid SARS‐CoV‐2 variant enzyme detection platform (SAVED) based on CRISPR‐Cas12a. It utilizes chimeric crRNA and short crRNA (15nt to 17nt nucleotide spacer) for SNP genotyping.…”

Section: Detection Of Vocs Using Crispr Systemmentioning

confidence: 99%

CRISPR‐Cas system: A promising tool for rapid detection of SARS‐CoV‐2 variants

Li,

Zhang,

Lin

et al. 2024

Journal of Medical Virology

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COVID‐19, caused by SARS‐CoV‐2, remains a global health crisis. The emergence of multiple variants with enhanced characteristics necessitates their detection and monitoring. Genome sequencing, the gold standard, faces implementation challenges due to complexity, cost, and limited throughput. The CRISPR‐Cas system offers promising potential for rapid variant detection, with advantages such as speed, sensitivity, specificity, and programmability. This review provides an in‐depth examination of the applications of CRISPR‐Cas in mutation detection specifically for SARS‐CoV‐2. It begins by introducing SARS‐CoV‐2 and existing variant detection platforms. The principles of the CRISPR‐Cas system are then clarified, followed by an exploration of three CRISPR‐Cas‐based mutation detection platforms, which are evaluated from different perspectives. The review discusses strategies for mutation site selection and the utilization of CRISPR‐Cas, offering valuable insights for the development of mutation detection methods. Furthermore, a critical analysis of the clinical applications, advantages, disadvantages, challenges, and prospects of the CRISPR‐Cas system is provided.

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“…The same principle of detecting cleaved reporters has been harnessed for other CRISPR and/or RNA-guided nucleases such as CRISPR-Cas12 or prokaryotic Argonaute proteins (16,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30).…”

Section: Introductionmentioning

confidence: 99%

New design strategies for ultra-specific CRISPR-Cas13a-based RNA-diagnostic tools with single-nucleotide mismatch sensitivity

Vargas

Osborn

Sinha

et al. 2023

Preprint

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The pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and new Cas13a variants for easier-to-implement Cas13-based diagnostics.

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Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a

Cited by 9 publications

References 38 publications

“Nano COVID-19”: Nanopore sequencing of spike gene to identify SARS-CoV-2 variants of concern

“Nano COVID-19”: Nanopore sequencing of spike gene to identify SARS-CoV-2 variants of concern

CRISPR‐Cas system: A promising tool for rapid detection of SARS‐CoV‐2 variants

New design strategies for ultra-specific CRISPR-Cas13a-based RNA-diagnostic tools with single-nucleotide mismatch sensitivity

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