MAGIK: A rapid and efficient method to create lineage-specific reporters in human pluripotent stem cells

Summary Precise insertion of a fluorescent protein into a lineage-specific gene in human pluripotent stem cells (hPSCs) presents challenges due to the low knockin efficiency and difficulties in selecting the correctly targeted cells. Here we introduce the ModRNA-based Activation for Gene Insertion and Knockin (MAGIK) approach to enhance knockin efficacy in hPSCs. MAGIK operates in two steps: first, it employs a Cas9-2A-p53DD modRNA with a mini-donor plasmid (without a drug-selection cassette) to significantly enhance efficiency; second, a dCas9 activator modRNA and a dgRNA are used to temporarily activate the successfully targeted gene, allowing for live cell sorting without single cell cloning. Consequently, MAGIK eliminates the need for drug selection cassettes or labor-intensive single cell colony screening, expediting precise genetic integration. We have demonstrated that MAGIK can be utilized to insert fluorescent proteins into various genes, including SOX17, NKX6.1, NKX2.5 and PDX1 , across multiple hPSC lines, showcasing its robustness. This innovative MAGIK approach streamlines the process and provides a promising solution for targeted genetic modifications in hPSCs.