A genome-scale CRISPR knockout library screen of THP-1 human macrophages was performed to identify loss-of-function mutations conferring resistance to uptake. The screen identified 183 candidate genes, from which 14 representative genes involved in actin dynamics (, , , , , , and ), glycosaminoglycan metabolism (), receptor signaling ( and ), lipid raft formation (), calcium transport ( and ), and cholesterol metabolism () were analyzed further. For some of these pathways, known chemical inhibitors could replicate the resistance phenotype, indicating their potential as targets for host-directed therapy. The screen indicated a role for the relatively uncharacterized gene in both invasion and macrophage differentiation. Upon differentiation, mutant macrophages were hyperinflammatory and did not exhibit characteristics typical of macrophages, including atypical morphology and inability to interact and phagocytose bacteria/particles. Immunoprecipitation confirmed an interaction of NHLRC2 with FRYL, EIF2AK2, and KLHL13. exploits macrophages to gain access to the lymphatic system and bloodstream to lead to local and potentially systemic infections. With an increasing number of antibiotic-resistant isolates identified in humans, infections have become major threats to public health. Therefore, there is an urgent need to identify alternative approaches to anti-infective therapy, including host-directed therapies. In this study, we used a simple genome-wide screen to identify 183 candidate host factors in macrophages that can confer resistance to infection. These factors may be potential therapeutic targets against infections.