STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells

• Published in: Cell reports methods Vol. 2; no. 10; p. 100300
• Main Authors: Blanch-Asensio, Albert, Grandela, Catarina, Brandão, Karina O., de Korte, Tessa, Mei, Hailiang, Ariyurek, Yavuz, Yiangou, Loukia, Mol, Mervyn P.H., van Meer, Berend J., Kloet, Susan L., Mummery, Christine L., Davis, Richard P.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 24.10.2022; Elsevier
• Subjects: Bxb1 integrase; cardiomyocyte; Cre recombinase; CRISPR-Cas9; disease modeling; human pluripotent stem cells; site-specific recombination; synthetic gene circuit; targeted gene modification; targeted gene modification; CRISPR-Cas9; disease modeling; cardiomyocyte; site-specific recombination; Bxb1 integrase; human pluripotent stem cells; synthetic gene circuit; Cre recombinase
• ISSN: 2667-2375; 2667-2375
• DOI: 10.1016/j.crmeth.2022.100300

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. [Display omitted] •Efficient and rapid platform to integrate DNA without size limits into the genome•Excision step leaves minimal scarring at the modified locus after the procedure•Technique expedites generating and evaluating multi-parameter reporter hiPSC lines•It also facilitates the simultaneous generation of panels of disease variant hiPSCs It is problematic to insert DNA fragments larger than ∼3 kb into a specific genomic locus in any mammalian cell, including hiPSCs. To address this, we have developed a platform that facilitates such site-specific insertion or replacement of genomic fragments with efficiencies that permit targeting of multiple constructs simultaneously. Compared with other approaches, STRAIGHT-IN demonstrably has virtually no restriction on the size of the DNA that can be inserted while maintaining precise control over modifications to the genome; it can thus significantly improve throughput in generating genetically modified cell lines. Blanch-Asensio et al. present a method to integrate large DNA payloads into specific genomic sites of hPSCs. This procedure offers a precise and rapid approach to generate genetically matched panels of hPSC lines with applications in research areas such as disease modeling and synthetic biology.