CRISPR-based nucleic acid diagnostics for pathogens

Yang, Hao; Zhang, Yong; Teng, Xianliang; Hou, Hongwei; Deng, Ruijie; Li, Jinghong

doi:10.1016/j.trac.2023.116980

Cited by 15 publications

(7 citation statements)

References 136 publications

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“…The sgRNA consists of a non-coding CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). This complex acts as a molecular scissor for targeted surveillance [86]. These technologies offer portable, highly sensitive tools for rapidly diagnosing infectious and noninfectious diseases.…”

Section: Crispr (Clustered Regularly Interspaced Short Palindromic Re...mentioning

confidence: 99%

Genomics for Emerging Pathogen Identification and Monitoring: Prospects and Obstacles

Vashisht,

Mondal

et al. 2023

BioMedInformatics

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Emerging infectious diseases (EIDs) pose an increasingly significant global burden, driven by urbanization, population explosion, global travel, changes in human behavior, and inadequate public health systems. The recent SARS-CoV-2 pandemic highlights the urgent need for innovative and robust technologies to effectively monitor newly emerging pathogens. Rapid identification, epidemiological surveillance, and transmission mitigation are crucial challenges for ensuring public health safety. Genomics has emerged as a pivotal tool in public health during pandemics, enabling the diagnosis, management, and prediction of infections, as well as the analysis and identification of cross-species interactions and the categorization of infectious agents. Recent advancements in high-throughput DNA sequencing tools have facilitated rapid and precise identification and characterization of emerging pathogens. This review article provides insights into the latest advances in various genomic techniques for pathogen detection and tracking and their applications in global outbreak surveillance. We assess methods that leverage pathogen sequences and explore the role of genomic analysis in understanding the epidemiology of newly emerged infectious diseases. Additionally, we address technical challenges and limitations, ethical and legal considerations, and highlight opportunities for integrating genomics with other surveillance approaches. By delving into the prospects and obstacles of genomics, we can gain valuable insights into its role in mitigating the threats posed by emerging pathogens and improving global preparedness in the face of future outbreaks.

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Section: Crispr (Clustered Regularly Interspaced Short Palindromic Re...mentioning

confidence: 99%

Genomics for Emerging Pathogen Identification and Monitoring: Prospects and Obstacles

Vashisht,

Mondal

et al. 2023

BioMedInformatics

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“…28 The dual recognition of Cas/crRNA to target ensures the specificity for SNV imaging. 29 The method has been used to quantify quinolone-resistant S. enterica strains and investigate the survival competition capacity of quinoloneresistant and quinolone-sensitive bacteria toward antimicrobial peptides.…”

Section: ■ Introductionmentioning

confidence: 99%

“…After specific amplification, we utilized a modified clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a to label the amplicon, thus enabling the specific binding of fluorophore-labeled DNA probes . The dual recognition of Cas/crRNA to target ensures the specificity for SNV imaging . The method has been used to quantify quinolone-resistant S.…”

Section: Introductionmentioning

confidence: 99%

In Situ Cas12a-Based Allele-Specific PCR for Imaging Single-Nucleotide Variations in Foodborne Pathogenic Bacteria

Liu,

Yang,

Liu

et al. 2024

Anal. Chem.

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In situ profiling of single-nucleotide variations (SNVs) can elucidate drug-resistant genotypes with single-cell resolution. The capacity to directly "see" genetic information is crucial for investigating the relationship between mutated genes and phenotypes. Fluorescence in situ hybridization serves as a canonical tool for genetic imaging; however, it cannot detect subtle sequence alteration including SNVs. Herein, we develop an in situ Cas12a-based amplification refractory mutation system-PCR (ARMS-PCR) method that allows the visualization of SNVs related to quinolone resistance inside cells. The capacity of discriminating SNVs is enhanced by incorporating optimized mismatched bases in the allele-specific primers, thus allowing to specifically amplify quinolone-resistant related genes. After in situ ARMS-PCR, we employed a modified Cas12a/CRISPR RNA to tag the amplicon, thereby enabling specific binding of fluorophore-labeled DNA probes. The method allows to precisely quantify quinolone-resistant Salmonella enterica in the bacterial mixture. Utilizing this method, we investigated the survival competition capacity of quinolone-resistant and quinolone-sensitive bacteria toward antimicrobial peptides and indicated the enrichment of quinolone-resistant bacteria under colistin sulfate stress. The in situ Cas12a-based ARMS-PCR method holds the potential for profiling cellular phenotypes and gene regulation with single-nucleotide resolution at the single-cell level.

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“…Due to the smaller size, Class 2 family Cas proteins have been widely deployed for revolutionary gene-editing tools and nucleic acid detection [16][17][18][19][20][21][22][23][24][25][26][27] . Although the application of the Class1 family may be limited by the factors such as multiple number of components, large size, and more complicated system, it also obtained extensive achievements in the field of gene editing and advancing the development of molecular diagnostic tools 6,16,24,[28][29][30][31][32][33][34][35] Type I CRISPR-Cas systems have garnered considerable attention due to their unique attributes and their potential applications in nucleic acid detection 36 . This system consists of six Cas proteins, comprising Cascade (Cas5, Cas6, Cas7, Cas8, Cas11), and the Cas3 nuclease (HD and HEL complex) 37 .…”

Section: Introductionmentioning

confidence: 99%

Repurpose Type I-A CRISPR-Cas3 for a Robust HPV Diagnosis

Hu,

et al. 2023

Preprint

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R-loop-triggered collateral single-stranded DNA (ssDNA) nuclease activity within Class 1 Type I CRISPR-Cas systems holds immense potential for nucleic acid detection. However, the hyperactive ssDNase activity of Cas3 introduces unwanted noise and false-positive results. In this study, we identified a novel Type I-A Cas3 variant derived from Thermococcus siculi, which remains in an auto-inhibited state until it is triggered by Cascade complex and R-loop formation. This Type I-A CRISPR-Cas3 system not only exhibits an expanded protospacer adjacent motif (PAM) recognition capability but also demonstrates remarkable intolerance towards mismatched sequences. Furthermore, it exhibits dual activation modes—responding to both DNA and RNA targets. The culmination of our research efforts has led to the development of the Hyper-Active-Verification Establishment (HAVE, 惠父). This innovation enables swift and precise human papillomavirus (HPV) diagnosis in clinical samples, providing a robust molecular diagnostic tool based on the Type I-A CRISPR-Cas3 system. Our findings contribute to understanding type I-A CRISPR-Cas3 system regulation and facilitate the creation of advanced diagnostic solutions with broad clinical applicability.

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CRISPR-based nucleic acid diagnostics for pathogens

Cited by 15 publications

References 136 publications

Genomics for Emerging Pathogen Identification and Monitoring: Prospects and Obstacles

Genomics for Emerging Pathogen Identification and Monitoring: Prospects and Obstacles

In Situ Cas12a-Based Allele-Specific PCR for Imaging Single-Nucleotide Variations in Foodborne Pathogenic Bacteria

Repurpose Type I-A CRISPR-Cas3 for a Robust HPV Diagnosis

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