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STRAIGHT-IN: a platform for rapidly generating panels of genetically modified human pluripotent stem cell lines

Nature Protocols (2024)Cite this article

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Abstract

Targeted integration of large DNA cargoes (>10 kb) or genomic replacements in mammalian cells, such as human pluripotent stem cells (hPS cells), remains challenging. Here we describe a platform termed serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation (STRAIGHT-IN) to circumvent this. First, a landing pad cassette is precisely inserted or used to replace specific genomic regions. The site-specific integrase Bxb1 then enables DNA constructs, including those >50 kb, to be integrated into the genome, while Cre recombinase excises auxiliary DNA sequences to prevent postintegrative silencing. Using a strategy whereby the positive selection marker is only expressed if the donor plasmid carrying the payload is correctly targeted, we can obtain 100% enrichment for cells containing the DNA payload. Procedures for expressing Cre efficiently also mean that a clonal isolation step is no longer essential to derive the required genetically modified hPS cells containing the integrated DNA, potentially reducing clonal variability. Furthermore, STRAIGHT-IN facilitates rapid and multiplexed generation of genetically matched hPS cells when multiple donor plasmids are delivered simultaneously. STRAIGHT-IN has various applications, which include integrating complex genetic circuits for synthetic biology, as well as creating panels of hPS cells lines containing, as necessary, hundreds of disease-linked variants for disease modeling and drug discovery. After establishing the hPS cell line containing the landing pad, the entire procedure, including donor plasmid synthesis, takes 1.5–3 months, depending on whether single or multiple DNA payloads are integrated. This protocol only requires the researcher to be skilled in molecular biology and standard cell culture techniques.

Key points

  • STRAIGHT-IN is a method for generating panels of genetically modified human pluripotent stem cell lines. Once a landing pad cassette is precisely inserted, the site-specific integrase Bxb1 enables DNA constructs, including those >50 kb, to be integrated into the genome, while Cre recombinase excises auxiliary DNA sequences.

  • This enables rapid and targeted integration of DNA payloads, irrespective of the size, and is achieved with minimal scarring.

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Fig. 1: Overview of LP and donor plasmid constructs, and genomic targeting of the LP.
Fig. 2: Workflow for generating STRAIGHT-IN acceptor hPS cell lines.
Fig. 3: Replacing genomic regions with the STRAIGHT-IN LP.
Fig. 4: Workflow for targeted integration of a donor plasmid carrying a DNA payload into the STRAIGHT-IN acceptor hPS cell line and subsequent enrichment.
Fig. 5: Workflow for Cre recombinase delivery and recovery of hPS cells with excised auxiliary sequences.
Fig. 6: Excising the auxiliary elements improves expression of the transgenes present in the DNA payload.
Fig. 7: ddPCR assays developed for determining LP copy number, quantifying donor plasmid integration and auxiliary element excision.
Fig. 8: Confluency of hPS cells.

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Data availability

The STRAIGHT-IN constructs used in this study are available from Addgene (https://www.addgene.org/Richard_Davis/). The datasets generated and analyzed in this study are provided as Source Data files. Source data are provided with this paper.

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Acknowledgements

We thank the LUMC hiPSC Hotel for co-ordinating the distribution of the Acceptor hiPS cell lines, N. Geijsen for providing Cas9 protein and F. Stewart for sharing plasmids and bacterial strains for recombineering. This work was supported by the Netherlands Organ-on-Chip Initiative, a Nederlandse Organisatie voor Wetenschappelijk Onderzoek Gravitation project funded by the Ministry of Education, Culture, and Science of the government of the Netherlands (024.003.001), a Starting Grant (STEMCARDIORISK; grant agreement no. 638030) from the European Research Council, a VIDI fellowship from the Netherlands Organization for Scientific Research (Nederlandse Organisatie voor Wetenschappelijk Onderzoek; ILLUMINATE; no. 91715303) and a ZonMw PSIDER consortium grant (no. 10250022120002; GREAT). The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW) is supported by a Novo Nordisk Foundation grant (NNF21CC0073729).

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Authors and Affiliations

  1. Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, the Netherlands

    Albert Blanch-Asensio, Catarina Grandela, Christine L. Mummery & Richard P. Davis

  2. The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Leiden University Medical Center, Leiden, the Netherlands

    Albert Blanch-Asensio, Christine L. Mummery & Richard P. Davis

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Contributions

A.B.-A. developed and optimized some of the protocols described in this manuscript, designed and performed the experiments, analyzed the data and wrote the manuscript. C.G. developed and optimized some of the protocols described in this manuscript, contributed to drafting the manuscript and revised it for important intellectual content. C.L.M. acquired some of the funding and revised the manuscript for important intellectual content. R.P.D. developed, designed and supervised the protocol, acquired some of the funding and wrote and revised the manuscript. All authors approved the final manuscript.

Corresponding author

Correspondence to Richard P. Davis.

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Competing interests

C.L.M. is a cofounder of Pluriomics BV (now Ncardia BV) and has advisory roles in HeartBeat.bio AG, Angios GmBH, Mogrify Limited and Sartorius AG. C.L.M. and R.P.D. declare research funding from Sartorius AG; however, this is for an unrelated study. A.B.-A. and C.G. declare no competing interests.

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Key references using this protocol

Blanch-Asensio, A. et al. Cell Rep. Methods 2, 100300 (2022): https://doi.org/10.1016/J.CRMETH.2022.100300

Blanch-Asensio, A. et al. Stem Cell Res. 66, 102991 (2023): https://doi.org/10.1016/j.scr.2022.102991

Supplementary information

Supplementary Information

Supplementary Figs. 1–3, Note, Procedures 1–5 and Table 1.

Supplementary Data 1

ddPCR source data for Supplementary Fig. 3.

Source data

Source Data Fig. 6

Flow cytometry and ddPCR source data.

Source Data Fig. 7

ddPCR source data.

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Blanch-Asensio, A., Grandela, C., Mummery, C.L. et al. STRAIGHT-IN: a platform for rapidly generating panels of genetically modified human pluripotent stem cell lines. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-01039-2

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