In the lytic cycle of Toxoplasma gondii, following successful invasion and replication, parasites eventually egress from the host cell. Parasite egress is not a passive process but a strictly orchestrated event driven by the interaction of factors derived from the host and parasites. The phylum Apicomplexa possesses secretory organelles called micronemes that release their contents: microneme proteins (MICs). MICs comprise various types of proteins, such as adhesins, proteases, and perforins, that are crucial for motility, invasion, and egress. In particular, perforin-like protein 1 (PLP1) is the single known MIC essential for both egress and virulence. However, it is incompletely understood whether other MICs also play pivotal roles during infection in vivo. In this study, we have generated a small-scaled gRNA library targeting approximately 300 T. gondii genes encoding proteins predicted to be localized at the apical complex (conoid and micronemes) and pellicles (IMC and plasma membrane) by hyperLOPIT. In vivo CRISPR screen analysis revealed various apical complex/pellicle-related genes that are important for parasite virulence. Among the top hits, we discovered that a novel microneme protein, whose function had been previously unknown, plays an essential role in the induction of egress by permeabilizing and disrupting both parasitophorous vacuole membrane and host plasma membrane. Moreover, we found that PLP1 alone is insufficient for parasite egress and that this novel MIC is also required. Collectively, our in vivo CRISPR screen provides a crucial clue to understanding the molecular mechanism of how MICs orchestrate parasite egress that contributes to the parasite in vivo adaptation and virulence.