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Related Concept Videos

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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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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Advancing Lateral Flow Detection in CRISPR/Cas12a Systems Through Rational Understanding and Design Strategies of

Irina V Safenkova1, Maria V Kamionskaya1, Dmitriy V Sotnikov1

  • 1A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia.

Biosensors
|December 24, 2025
PubMed
Summary

Optimizing CRISPR/Cas12a-lateral flow tests (LFTs) by adjusting interaction time and component concentrations significantly reduces false positives. This enhances sensitivity for rapid, instrument-free nucleic acid diagnostics.

Keywords:
CRISPR/Cas12aErwinia amylovoralateral flow strippoint-of-care testingreportertrans-target

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Diagnostics

Background:

  • CRISPR/Cas12a systems combined with lateral flow tests (LFTs) offer rapid, instrument-free nucleic acid diagnostics.
  • Conventional CRISPR/Cas12a-LFT formats can suffer from false-positive signals and sensitivity issues due to component interactions and kinetics.

Purpose of the Study:

  • To experimentally and theoretically analyze limitations in CRISPR/Cas12a-LFT systems.
  • To define practical solutions for improving the reliability and sensitivity of these diagnostic platforms.

Main Methods:

  • Evaluated 480 LFT configurations, varying component concentrations and interaction times.
  • Employed a mathematical model to analyze LFT kinetics.
  • Tested strategies including pre-incubation, reduced reporter concentration, and optimized nanoparticle/reporter architecture.

Main Results:

  • Insufficient interaction time was the primary cause of false positives; 5 min pre-incubation eliminated artifacts.
  • Lowering reporter concentration to 20 nM and using smaller gold nanoparticles with multivalent reporters improved sensitivity.
  • Optimized configurations achieved over 50-fold sensitivity improvement and detected DNA targets down to 20 pM.

Conclusions:

  • Identified key strategies to eliminate false positives and enhance sensitivity in CRISPR/Cas12a-LFT diagnostics.
  • The developed strategies are general and applicable to other DNA targets and amplification-free diagnostic assays.