Virginia Research Day 2025

Medical Student Research Biomedical

17 Validation Study Using DNA Extraction and PCR of Different Cell Culture Lines to Verify Gene Sequences for Future CRISPR Application

Erin Onken; Priyanka Avasarala; Ciera Miller; Nicole Wilson; Ramu Anandakrishnan, PhD Corresponding author: eonken@vcom.edu

Edward Via College of Osteopathic Medicine Center for One Health Research

CRISPR-based genome editing has revolutionized molecular biology by enabling precise and efficient modifications of genetic material, offering promising applications in both research and therapeutic fields. However, the success of CRISPR interventions hinges on the accurate validation of target gene sequences within selected cell lines, as sequence integrity is essential for ensuring specificity and minimizing off-target effects. This study presents a systematic validation of gene sequences in various cell culture lines through DNA extraction and polymerase chain reaction (PCR) analysis, laying the groundwork for future CRISPR-based applications. The main

objective was to verify the accuracy and integrity of target gene sequences, a critical step in ensuring the success of CRISPR genome editing. Multiple cell lines were cultured, and genomic DNA was isolated using standardized extraction protocols. PCR amplification was employed to selectively amplify specific gene regions of interest. The amplified DNA fragments were analyzed for selected genes of interest. Comparisons were made with knockout (KO) gene cell lines to further verify the specificity of the target genes. KO cell lines, which lack the functional gene of interest, served as negative controls in the PCR assays. The absence of amplified products in KO

lines confirmed the effectiveness of the primers and ensured that the observed PCR products in wild-type lines were indeed specific to the target gene. This comparative analysis enhanced the validation process by ruling out potential nonspecific amplification. The validation established a reliable framework for identifying sequence anomalies and confirmed that the selected cell lines possess the correct gene sequences required for precise and effective CRISPR interventions. This approach minimizes off-target effects and ensures greater accuracy in future gene editing experiments.

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