Fig 1: COX4I1 governs pivotal stages of Complex IV assembly. A) Schematic depiction of the COX4I1 high‐resolution CRISPR tiling library screen conducted in Molm13‐Cas9+ cells. B) 2D annotation illustrating COX4I1 peptide position (x‐axis) and CRISPR tiling score (y‐axis). The red line represents the smoothed model of the CRISPR scan score derived from individual sgRNAs (dots) targeting the coding exons of COX4I1. The CRISPR hypersensitive P1 and P2 regions are indicated. C,D) Impact of wild‐type (WT), ΔP1, and ΔP2 COX4I1 expression on (C) cell number expansion and (D) mitochondrial respiration in COX4I1‐KO Molm13 cells (n = 3 for each group). E) Western blot analysis of COX4I1, COX5A, MT‐CO2, COX6B1, and β‐actin in COX4I1‐KO Molm13 cells transduced with WT, ΔP1, and ΔP2 COX4I1. F,G) Volcano plots comparing mitochondrial protein abundances in COX4I1‐KO Molm13 cells transduced with (F) ΔP1 versus WT COX4I1 and (G) ΔP2 versus WT COX4I1. H) Comparative analysis of the impact of ΔP1 (x‐axis) and ΔP2 (y‐axis) COX4I1 on the mitochondrial proteome (dots). Dashed lines denote a 10‐fold difference between ΔP1 and ΔP2. COX4I1‐regulated mitochondrial proteins defined in Figure 4F are highlighted with (F) blue dots, (G) green dots, and (H) red dots. I) Human Complex IV structural model (PDB 5Z62) colored by COX1 module (pink), COX2 module (orange), and COX3 module (blue). The P1, P2, and transmembrane (TM) regions of COX4I1 are highlighted in red. J) Network model illustrating critical interactions (black lines) between COX4I1 and other components of Complex IV. Data are presented as mean ± SEM. * p < 0.01 by two‐sided Student's t‐test.