Optimized CRISPR Perturbation Illuminates the Functions of RNA Modification Proteins
iMAP is a large-scale CRISPR perturbation platform based on a germline-transmitted transgene comprising individually floxed, tandemly linked guides that can be inducibly expressed via Cre-mediated recombination. A key limitation was strong recombination bias favoring the proximal guides. Here, we effectively mitigated this bias by inactivating a recombination hotspot, yielding guide representation nearly as uniform as traditional lentiviral libraries. Applying this optimized platform, we systematically profiled 70 (52%) RNA modification factors across 46 mouse tissues, uncovering pervasive tissue-specific essentiality. Notably, iMAP displayed much higher essential gene recovery than prior lentiviral CRISPR screens, attributable to its stronger guide depletion and lower technical noise. Finally, in a tumor model, iMAP identified Thg1l as a repressor of NK cell TNF production; its ortholog knockout enhanced human NK cell-mediated tumor killing, underscoring therapeutic potential. All data are available in an interactive iMAP database. This work advances the Perturbation Atlas, a foundational resource for functional genomics. TeaserA unique CRISPR perturbation tool has been upgraded and deployed to reveal functions of epigenetic regulators in mice.