@article{81bea03064044d8f875cfa6c9facc999,
title = "STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells",
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.",
keywords = "Bxb1 integrase, cardiomyocyte, Cre recombinase, CRISPR-Cas9, disease modeling, human pluripotent stem cells, site-specific recombination, synthetic gene circuit, targeted gene modification",
author = "Albert Blanch-Asensio and Catarina Grandela and Brand{\~a}o, {Karina O.} and {de Korte}, Tessa and Hailiang Mei and Yavuz Ariyurek and Loukia Yiangou and Mol, {Mervyn P.H.} and {van Meer}, {Berend J.} and Kloet, {Susan L.} and Mummery, {Christine L.} and Davis, {Richard P.}",
note = "Funding Information: We thank M. Bellin for providing the control (LUMC0020iCTRL) hiPSC line, M. de Graaf and L. Voortman (LUMC Light Microscopy Facility) for microscopy assistance, the LUMC flow cytometry facility for sorting the cells, and the Laboratory for Diagnostic Genome Analysis (LUMC) for karyotyping. We also acknowledge Francis Stewart for sharing plasmids for recombineering and Niels Geijsen for providing the Cas9 protein. Some panels within figures were created with BioRender.com . This work was supported by a Starting Grant (STEMCARDIORISK; grant agreement no. 638030 ) and a Proof of Concept grant (ACQUIRE; grant agreement no. 885469 ) from the European Research Council (ERC) under the European Union{\textquoteright}s Horizon 2020 Research and Innovation Program; a VIDI fellowship from the Netherlands Organization for Scientific Research ( Nederlandse Organisatie voor Wetenschappelijk Onderzoek NWO; ILLUMINATE; no. 91715303 ); the Netherlands Organ-on-Chip Initiative, an NWO Gravitation project funded by the Ministry of Education , Culture, and Science of the government of the Netherlands (024.003.001); and by a Novo Nordisk Foundation grant ( NNF21CC0073729 ; reNEW). Funding Information: We thank M. Bellin for providing the control (LUMC0020iCTRL) hiPSC line, M. de Graaf and L. Voortman (LUMC Light Microscopy Facility) for microscopy assistance, the LUMC flow cytometry facility for sorting the cells, and the Laboratory for Diagnostic Genome Analysis (LUMC) for karyotyping. We also acknowledge Francis Stewart for sharing plasmids for recombineering and Niels Geijsen for providing the Cas9 protein. Some panels within figures were created with BioRender.com. This work was supported by a Starting Grant (STEMCARDIORISK; grant agreement no. 638030) and a Proof of Concept grant (ACQUIRE; grant agreement no. 885469) from the European Research Council (ERC) under the European Union's Horizon 2020 Research and Innovation Program; a VIDI fellowship from the Netherlands Organization for Scientific Research (Nederlandse Organisatie voor Wetenschappelijk Onderzoek NWO; ILLUMINATE; no. 91715303); the Netherlands Organ-on-Chip Initiative, an NWO Gravitation project funded by the Ministry of Education, Culture, and Science of the government of the Netherlands (024.003.001); and by a Novo Nordisk Foundation grant (NNF21CC0073729; reNEW). Conceptualization, C.G. A.B.-A. and R.P.D.; methodology, C.G. A.B.-A. and R.P.D.; software, H.M. and B.J.v.M.; formal analysis, A.B.-A. T.d.K. H.M. and R.P.D.; investigation, C.G. A.B.-A. K.O.B. T.d.K. Y.A. L.Y. and M.P.H.M.; resources, S.L.K.; data curation, A.B.-A. and H.M.; writing – original draft, R.P.D. and A.B.-A.; writing – review & editing, all authors; supervision, R.P.D.; funding acquisition, C.L.M. and R.P.D. C.L.M. is a cofounder of Pluriomics B.V. (now Ncardia B.V.) and has advisory roles in Sartorius AG, Mogrify Limited, and Angios GmBH. C.L.M. and R.P.D. declare research funding from Sartorius AG; however, this is for an unrelated study. All other authors declare no potential conflicts of interest. Funding Information: C.L.M. is a cofounder of Pluriomics B.V. (now Ncardia B.V.) and has advisory roles in Sartorius AG, Mogrify Limited, and Angios GmBH. C.L.M. and R.P.D. declare research funding from Sartorius AG; however, this is for an unrelated study. All other authors declare no potential conflicts of interest. Publisher Copyright: {\textcopyright} 2022 The Author(s)",
year = "2022",
month = oct,
day = "24",
doi = "10.1016/j.crmeth.2022.100300",
language = "English",
volume = "2",
journal = "Cell Reports Methods",
issn = "2667-2375",
publisher = "Cell Press",
number = "10",
}