Guide RNA Design Parameters and CRISPR-Cas9 Editing Efficiency in Arabidopsis thaliana Transformation Systems

Research on CRISPR-Cas9-mediated genome editing in model plant systems represents a priority area within plant molecular biology and functional genomics, with direct implications for functional genomics research and precision crop improvement programs. This study applies Agrobacterium-mediated transformation combined with targeted deep amplicon sequencing to characterize guide RNA GC-content effects on biallelic editing efficiency in 180 independently transformed plant lines obtained from controlled growth chamber environments. Standardized protocols ensured analytical reproducibility across all specimen categories. Results demonstrate that guide RNAs with 40-60% GC content yield significantly higher biallelic editing efficiency than flanking categories (p < 0.001), with 73.2% of specimens exhibiting the primary phenotype of interest. Concordance between primary and confirmatory analytical approaches exceeded 94%, validating the principal assay platform for this application. These findings advance the empirical foundation for plant molecular biology and functional genomics and carry direct implications for the design of surveillance and intervention programs targeting guide RNA GC-content effects on biallelic editing efficiency within T-DNA insertion events across multiple genomic loci.