STRAIGHT-IN: A platform for high-throughput targeting of large DNA payloads into human pluripotent stem cells

Inserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease-associated variants for precision medicine initiatives would greatly benefit from tools that facilitate this process. Here, we merge the strengths of different classes of site-specific recombinases and combine these with CRISPR/Cas9-mediated homologous recombination to develop a strategy for stringent site-specific replacement of genomic fragments at least 50 kb in size in human induced pluripotent stem cells (hiPSCs). We demonstrate the versatility of STRAIGHT-IN (Serine and Tyrosine Recombinase Assisted Integration of Genes for High-Throughput INvestigation) by: (i) inserting various combinations of fluorescent reporters into hiPSCs to assess excitation-contraction coupling cascade in derivative cardiomyocytes, and; (ii) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorder into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically-matched panels of hiPSC lines efficiently and cost-effectively.