Fat1 Knockout cell line (ID8 )

Adopting Red Cotton CRISPR Gene Editing Designer (www.rc-crispr.com), two sgRNAs targeting the Fat1 gene are designed to generate a knockout strategy. The two sgRNA sequences are subsequently cloned into the EZ-editor™ vector and introduced into ID8 cells via electroporation or lentiviral transduction. Single-cell clones are then generated using the limiting dilution method. Genomic DNA from individual clones is subjected to nucleic acid lysis and PCR amplification using the EZ-editor™ Monoclone Genotype Validation Kit (Cat# YK-MV-1000). The edited loci are further verified by Sanger sequencing to confirm the genotype. After secondary validation and quality confirmation, Fat1 Knockout cell line (ID8 ) is expanded and cryopreserved for downstream applications.