Antimicrobial resistance (AMR) in Gram-negative bacteria poses an urgent public health challenge. While traditional approaches have identified essential genes through complete disruption, understanding how partial loss of function affects bacterial survival has remained largely intractable. This thesis applies CRISPR interference (CRISPRi) technology to systematically examine essential gene function in Gram-negative pathogens, focusing on three areas: mapping essential gene vulnerabilities in Acinetobacter baumannii, characterizing hostspecific essential gene requirements in Pseudomonas aeruginosa, and comparing essential gene networks across Enterobacterales species. The systematic analysis identified essential genes and pathways that demonstrate acute sensitivity to knockdown, providing promising therapeutic targets. In A. baumannii, this work demonstrated genes and pathways that modulate β-lactam sensitivity, establishing an unexpected link between NADH dehydrogenase activity and polymyxin sensitivity. In P. aeruginosa, approaches to overcome infection bottlenecks identified 178 genes with increased vulnerability in the host environment. Comparative analysis of essential gene networks across Escherichia coli, Enterobacter cloacae, and Klebsiella pneumoniae characterized both shared vulnerabilities and species-specific differences in antibiotic response. These findings challenge binary classifications of gene essentiality and demonstrate that many promising drug targets may have been overlooked by structural approaches like transposon sequencing and gene deletion libraries. By enabling controlled knockdown rather than complete deletion, CRISPRi establishes fundamental patterns of bacterial adaptation and expands opportunities for therapeutic development.