STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells
Albert Blanch-Asensio, Catarina Grandela, Karina O. Brandão, Tessa de Korte, Hailiang Mei, Yavuz Ariyürek, Loukia Yiangou, Mervyn P.H. Mol, Berend J. van Meer, Susan L. Kloet, Christine L. Mummery, Richard P. Davis
Abstract
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 (1) inserting various combinations of fluorescent reporters into hiPSCs to assess the excitation-contraction coupling cascade in derivative cardiomyocytes and (2) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorders into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically matched panels of hiPSC lines efficiently and cost effectively.