Optimized CRISPR Perturbation Illuminates the Functions of RNA Modification Proteins

we have previously developed an in situ CRISPR screening platform in mice named iMAP, where Cas9 guides are stored in germline-transmitted transgenes but inducibly released (expressed) via Cre-mediated recombination. A major limitation of iMAP is a strong bias in guide representation following the recombination. We now report that this bias was effectively mitigated by eliminating a recombination hotspot at the transgene. We next profiled, across 46 tissues, the abundance of the guides targeting 71 (52%) genes encoding writers, readers and erasers of RNA modifications, revealing their context-specific functions. Furthermore, we identified, using a mouse tumor model, several candidate regulators of mouse NK cell activation, and found that their ortholog knockout boosted human NK cells, thus offering potential targets for improving cancer immunotherapy. Finally, a public database was established for accessing and analyzing iMAP data. Our study marks an important step toward the creation of the Perturbation Atlas.