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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...

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Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format
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Systematically Investigating CRISPR/Cas12a Fluorescent Biosensor for Sensitive and Specific Single Nucleotide

Zhenlin Shang1, Sitong Liu1, Dongxu Liu1

  • 1School of Light Industry Science and Engineering, Beijing Technology and Business University, Beijing, 100048, P. R. China.

Journal of Fluorescence
|May 22, 2025
PubMed
Summary

This study developed a CRISPR/Cas12a system with optimized crRNAs and molecular beacon reporters for precise single nucleotide variation (SNV) detection. The method accurately identifies SARS-CoV-2 mutations like D614G and N501Y, enabling rapid pathogen detection.

Keywords:
CRISPR/Cas12aD614GN501YSingle-nucleotide variations

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Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases
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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • Accurate detection of single nucleotide variations (SNVs) is crucial for diagnosing invasive diseases, identifying pathogens, and predicting drug responses.
  • Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants, such as D614G and N501Y, exhibit increased infectivity, highlighting the need for rapid mutation detection methods.

Purpose of the Study:

  • To develop and optimize a CRISPR/Cas12a-based system for the sensitive and specific detection of SNVs, using SARS-CoV-2 variants as a model.
  • To enhance detection specificity by optimizing CRISPR RNA (crRNA) design and exploring different fluorescent reporters.

Main Methods:

  • Utilized CRISPR/Cas12a technology with three types of fluorescent reporters and two crRNAs for SNV detection.
  • Systematically screened crRNA base substitutions to identify optimal positions for enhanced specificity.
  • Investigated the specificity of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and molecular beacon (MB) reporters.
  • Engineered lateral flow strips for visual, user-friendly result presentation.

Main Results:

  • Identified that the middle position of the crRNA significantly enhances specificity for SNV detection.
  • Demonstrated that molecular beacon (MB) fluorescent reporters provide superior discriminatory factors compared to ssDNA and dsDNA reporters.
  • Confirmed the system's effectiveness with SARS-CoV-2 Omicron variants, showcasing its potential for clinical applications.
  • Successfully engineered lateral flow strips for naked-eye visualization of results.

Conclusions:

  • The developed CRISPR/Cas12a system offers a powerful and adaptable tool for monitoring key mutations in pathogens.
  • The method demonstrates high specificity and sensitivity for SNV detection, with potential for modification to detect emerging variants.
  • The integration of lateral flow strips facilitates rapid, point-of-care diagnostics.