STRAIGHT-IN Dual: a platform for dual single-copy integrations of DNA payloads and gene circuits into human induced pluripotent stem cells.

Targeting DNA payloads into human induced pluripotent stem cells (hiPSCs) typically requires multiple inefficient steps, slowing the testing of gene circuits and cell-fate programmes. Here we show that STRAIGHT-IN Dual enables simultaneous, allele-specific, single-copy integration of two DNA constructs efficiently within 1 week. STRAIGHT-IN Dual leverages the STRAIGHT-IN platform for near-scarless payload integration, facilitating the recycling of components for further modifications. Using STRAIGHT-IN Dual, we investigate how promoter choice and gene syntax influence transgene silencing and how these design features affect reporter expression and forward programming of hiPSCs into neurons, motor neurons and endothelial cells. We also incorporate a grazoprevir-inducible synthetic gene switch that complements tetracycline-inducible control, providing tunable and temporally controlled expression of different transcription factors within the same cell. STRAIGHT-IN Dual generates homogeneous engineered hiPSC populations, accelerating synthetic biology design-build-test cycles in stem cells and enabling controlled comparisons of circuit performances.