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Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

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Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents
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Published on: August 16, 2024

Thermodynamically programmed one-pot CRISPR platform for point-of-care SNP genotyping.

Xiaolong Wu1, Yanan Li1, Yumeng Cao1

  • 1Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Kowloon, Hong Kong, China.

Nature Communications
|July 7, 2026
PubMed
Summary
This summary is machine-generated.

This study introduces Thermodynamically Encoded Molecular Programming for One-pot diagnostics (TEMPO), a novel CRISPR diagnostic method. TEMPO uses DNA primer design to control reaction timing, enabling sensitive and rapid pathogen detection and genotyping.

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Last Updated: Jul 9, 2026

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Published on: April 8, 2017

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Diagnostics

Background:

  • One-pot CRISPR diagnostics face challenges due to the incompatibility between rapid nucleic acid amplification and CRISPR activation.
  • CRISPR activation consumes amplification substrates, destabilizing reaction kinetics and limiting diagnostic efficiency.

Purpose of the Study:

  • To develop a method for programming reaction order in one-pot CRISPR diagnostics through thermodynamic design of DNA primers.
  • To overcome the kinetic instability in isothermal amplification and CRISPR activation for improved diagnostic performance.

Main Methods:

  • Designed DNA primers with varying binding strengths to create sequential amplification stages, delaying CRISPR activation.
  • Integrated the protospacer adjacent motif (PAM) into primers, enhancing target accessibility and maintaining single-nucleotide discrimination.
  • Utilized an ordinary differential equation model to predict and optimize primer design for threshold behavior.

Main Results:

  • Achieved attomolar sensitivity within 30 minutes using the developed TEMPO system.
  • Demonstrated sequencing-concordant single nucleotide polymorphism (SNP) genotyping.
  • Successfully detected pathogens in a single-step microfluidic format.

Conclusions:

  • Thermodynamic primer design enables precise control over reaction kinetics in one-pot CRISPR diagnostics.
  • TEMPO offers a robust and sensitive platform for rapid molecular detection and genotyping.
  • This approach expands the applicability of CRISPR-based diagnostics to diverse targets and clinical settings.