Abstract 2502 Development of a Molecular Cloning Strategy for Validating CRISPR-Mediated Gene Editing

Our laboratory has shown that the type I interferon-induced human protein Schlafen 11 (SLFN11) functions as a restriction factor for flaviviruses and HIV-1 in in vitro cultured cells.To explore the role of this innate immune pathway in vivo, we plan to evaluate the activity of the SLFN11 mouse orthologs, SLFN8 and SLFN9, in restricting the flavivirus West Nile virus in a mouse model.Before conducting in vivo experiments, we aim to determine whether these mouse proteins can block West Nile virus infection in cultured cells.To achieve this, we utilized the CRISPR/Cas9 system to knock out SLFN8 and SLFN9 expression.One major challenge in validating the effectiveness of this approach is the lack of available antibodies against these proteins.To circumvent this, we developed a cloning strategy to verify the sequence of the endogenous Slfn8 and Slfn9 genes at the CRISPR/Cas9-targeted regions.We isolated genomic DNA from CRISPR/Cas9-treated cells and performed PCR amplification of a DNA fragment flanking the gene region containing the CRISPR/Cas9 target site.The resulting PCR product was cloned into the polylinker region of the pUC19 plasmid and transformed into E. coli.These bacteria were then plated on LB agar containing ampicillin and the Beta-galactosidase substrate, X-Gal.Colonies carrying pUC19 plasmids with disrupted Beta-galactosidase genes-due to the successful insertion of the PCR product-remained white, whereas colonies with intact plasmids turned blue.Plasmids isolated from white colonies were confirmed to contain the PCR insert through restriction digest analysis, and the sequence of the insert was determined.This process enabled us to select an SLFN8 knock-out cell line, demonstrating that our strategy is effective even when antibodies against the target protein are unavailable.This robust approach will allow us to move forward with the in vitro studies, providing critical insights into the role of SLFN8 and SLFN9 in the innate immune response to West Nile virus.