Reaction Kinetics of CRISPR trans-Cleavage Controlled Using Isotachophoresis.

Area of Science:

• Biotechnology
• Molecular Biology
• Biophysics

Background:

• CRISPR-based diagnostics offer high specificity for nucleic acid detection.
• Assay sensitivity is often limited by slow CRISPR *trans*-cleavage kinetics (0.1-1 turnover/sec).

Purpose of the Study:

• To analyze and accelerate CRISPR *trans*-cleavage kinetics using electric-field-driven isotachophoresis (ITP).
• To develop a quantitative framework for optimizing CRISPR-based assays.

Main Methods:

• Developed a reaction-transport model coupling ITP focusing, mixing, and preconcentration with CRISPR kinetics.
• Identified key kinetic regimes and derived analytical approximations for ITP-enhanced CRISPR reactions.
• Validated model predictions through experimental studies across various target concentrations.

Main Results:

• Predicted a 10- to 100-fold reduction in CRISPR reaction duration using ITP compared to standard assays.
• Demonstrated experimental validation of ITP's acceleration effect on CRISPR *trans*-cleavage.
• Established a quantitative framework for understanding ITP-CRISPR interactions.

Conclusions:

• Electric-field-driven ITP significantly accelerates CRISPR *trans*-cleavage kinetics.
• ITP offers a viable strategy for enhancing the speed and sensitivity of CRISPR-based diagnostic assays.
• Provides guidance for designing future electric-field-mediated nucleic acid detection systems.