Extraintestinal E. coli have adapted to survive in secondary environments outside of the intestines of a host. The genetically diverse phylogroup D was use to validate two previous GWAS analysis that identified single nucleotide variants to have a positive or negative effect on biofilm formation. We selected ten variants from TreeWAS based on current literature to identify patterns among a new set of E. coli isolates, to identify SNV that can be used as genetic markers for biofilm formation. DBGWAS was used to predict and validate the predicted value of a SNV, by conducting an allelic exchange by using a CRISPR/Cas9 system, pCAGO, that allowed for scarless genome editing. This allelic exchange and deletion of the gene led to no statistical significance when biofilm formation and growth rate were tested. This led to variation in biofilm formation but didn’t affect the phenotype in a statistically significant manner.