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

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.