Quantitative Structural Characterization of CRISPR-Cas Protein–Nucleic Acid Interactions Through Image-Based Analysis
Keywords:
CRISPR–Cas systems, Electrophoretic mobility shift assay, Protein–nucleic acid interactions, Band densitometry, Molecular recognitionAbstract
Electrophoretic mobility shift assays (EMSAs) provide a valuable approach for investigating protein–nucleic acid interactions and characterizing molecular complexes associated with CRISPR–Cas systems. The electrophoretic band distributions under control, mutant, mutant with Cas11, wild-type (WT) and crRNA conditions were quantified and evaluated in this study. Five sets of EMSA gel images, each containing 50 electrophoretic lanes with 100 band intensities were examined. The analytical method used was lane identification, background correction, pre-integration of the intensity of the bands, and normalization within the lane to obtain relative signal distributions. In the control condition, two band regions (22.34%, 77.66%) of normalized intensity were observed while in the mutant (Cas11 condition), two band regions (43.93%, 56.07%) of normalized intensity were observed. In the mutant condition, there was only one band region exhibited and in the WT there were three band regions exhibited with 7.87%, 32.26% and 59.87% respectively. The condition of the crRNA exhibited two band areas, 31.53% and 68.47%, with the difference in electrophoretic signal distribution. Comparative evaluation showed the distinct pattern of band dominance, electrophoretic heterogeneity and variability in the experimentation conditions in the lanes. The findings in this study illustrate the utility of systematic characterization of CRISPR-associated electrophoretic profiles using quantitative EMSA densitometry as a foundation for further studies of molecular recognition, protein–nucleic acid interactions, and protein organization of the CRISPR-associated complexes.
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