In vitro validation of a CRISPR/Cas9 guide RNA targeting COL1A1 gene by cleavage assay.

The CRISPR/Cas9 system has become a widely used tool for genome editing; however, editing efficiency is largely determined by the design and functional activity of the guide RNA (gRNA). Experimental validation prior to cellular applications is particularly important when targeting genes associated with genetic disorders, such as COL1A1, which is frequently mutated in Osteogenesis Imperfecta. This study performed an in vitro functional validation of a CRISPR/Cas9 gRNA designed to target exon 2 of the COL1A1 gene, using a cell-free cleavage assay as a preliminary step before cellular genome editing applications. Three gRNAs targeting distinct COL1A1 exons were designed using bioinformatics tools; the exon 2 candidate was selected for experimental validation based on predicted efficiency, favorable off-target profile, and successful PCR amplification of its target region. The selected gRNA was assembled with recombinant Cas9 protein into a ribonucleoprotein complex, which was incubated with PCR-amplified DNA substrates derived from five distinct human cell lines. Cleavage products were resolved by polyacrylamide gel electrophoresis and quantified by densitometric analysis. The gRNA directed reproducible, specific Cas9-mediated cleavage at the intended COL1A1 exon 2 target site across all cell lines tested, with mean cleavage efficiencies ranging from 58.79% to 68.84%. These results confirm functional validation of the gRNA through in vitro cleavage activity and support its candidate selection for subsequent cellular studies targeting COL1A1, representing an initial step toward future knockout-style cellular models of Osteogenesis Imperfecta.