Quantitative Analysis of CRISPR Encoding Plasmid DNA-Nanocarrier Interactions by Gel Electrophoresis and Densitometry.

Nanoparticle-based carriers are key systems for the transport and delivery of nucleic acids in gene therapy, RNA therapeutics, and genome editing applications. The development of efficient delivery systems for CRISPR-Cas technologies requires reliable methods to evaluate nanocarrier-nucleic acid interactions, binding efficiency, and loading capacity. Agarose gel electrophoresis is commonly used to study these interactions through gel retardation assays, but results are often interpreted qualitatively based on visual inspection of DNA band migration. In this chapter, we present a quantitative approach that combines agarose gel electrophoresis with densitometric analysis of plasmid DNA (pDNA) bands to evaluate the interaction between pDNA and inorganic nanocarriers. The method relies on quantifying the fraction of free pDNA that migrates through the gel, while nanocarrier-pDNA systems remain near the loading wells. Band intensities are analyzed using ImageJ software to determine the amount of free pDNA and to estimate binding efficiency through depletion analysis. This protocol provides a simple and accessible strategy to quantify the loading of pDNA encoding CRISPR components, guide RNA constructs, or other nucleic acid cargos, facilitating the development and optimization of nanoparticle-based delivery systems for genome editing applications.