Biofilm infections on indwelling biomedical devices are the leading cause of global hospital infections. Among the most common are catheter-associated infections caused by the bacteria Escherichia coli and Staphylococcus aureus that adhere to the device surface. These infections are typically treated using broad-spectrum antibiotics, which can promote antibiotic resistance and often are not fully effective. Towards preventing these infections, mechanistic knowledge of the genes and pathways controlling bacterial adhesion on relevant physicochemically diverse biomaterials is limited yet could be used for the rational design of targeted anti-biofouling agents to prevent infections on biomedical device surfaces. Our goal in this research was to elucidate how the genetic contributions to cellular adhesion are affected by the physicochemical properties of the biomaterial surface for clinically relevant bacterial strains. Due to its targeted repression and simple design rules, CRISPR interference (CRISPRi) is an ideal choice for genome-wide investigation of gene expression associated with complex phenotypes such as cell adhesion. However, genetic tools, including CRISPRi, have primarily been developed in model laboratory strains of select bacteria with limited knowledge on the transferability to non-model clinical strains. Thus, the aims of this research were to establish genetic parts and CRISPRi tools for non-model bacterial strains (S. aureus ATCC 12600, E. coli Nissle 1917, E. coli CFT073, and E. coli UMN026) and to investigate the effects of gene repression across the genome on cellular adhesion to physicochemically diverse biomaterials. In Aim 1, we created the first toolbox of characterized genetic parts for the bacterium S. aureus to precisely control gene expression and enable further study of its mechanisms for cellular adhesion. We demonstrated use of these genetic parts to rationally design and optimize a genetically-encoded biosensor. In Aim 2, we studied and optimized three CRISPRi systems for four E. coli strains, ranging from a laboratory strain MG1655 to three non-model strains, to understand the strain-specific design rules for this approach. Data analysis revealed high transferability in terms of relative gene regulation but key strain-specific differences in both absolute levels of gene regulation and cellular growth toxicity, two important considerations for applications of CRISPRi. In Aim 3, we developed and implemented a comprehensive, genome-wide CRISPRi library for E. coli MG1655 in multiplexed adhesion assays for hydrophilic polyethylene glycol and hydrophobic polydimethylsiloxane biomaterials of varying stiffnesses to determine how hydrophilicity and stiffness affect gene fitness for cell adhesion to each surface. Results revealed greater correlations between biomaterials of the same chemistry rather than stiffness, along with more gene hits that reduced cell adhesion to all biomaterials as compared to those that increased adhesion. Notably, our findings revealed many novel genes that control cell adhesion to these biomaterials. These results increase our knowledge of this complex cellular process and could be used to identify genetic targets for the design of effective targeted anti-biofouling agents, such as silencing oligonucleotides, to prevent biofilm infections. Additionally, the platform for genome-wide CRISPRi selections developed in this study could be broadly applied to other selective conditions or used in other bacteria, including non-model strains.