Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells

Abstract

Selection of the target site is an inherent question for any project aiming for directed transgene integration. Genomic safe harbour (GSH) loci have been proposed as safe sites in the human genome for transgene integration. Although several sites have been characterised for transgene integration in the literature, most of these do not meet criteria set out for a GSH and the limited set that do have not been characterised extensively. Here, we conducted a computational analysis using publicly available data to identify 25 unique putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression and minimal disruption of the native transcriptome in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.

Data availability

Unprocessed RNAseq FASTQ files generated for this study will be available from ENA: PRJEB49564 accession numbers: ERS16364945-ERS16364998.Custom computational scripts used for the GSH search will be available from https://github.com/foo-labHigh content imaging data will be available on Dryad.All other data generated during this study are included in the manuscript and supporting file

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Matias I Autio

    Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore
    For correspondence
    autiomi@gis.a-star.edu.sg
    Competing interests
    Matias I Autio, Patent application PCT/SG2022/050888.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9579-9617
  2. Efthymios Motakis

    Cardiovascular Disease Translational Research Programme, National University of Singapore, Singapore, Singapore
    Competing interests
    Efthymios Motakis, Patent application PCT/SG2022/050888.
  3. Arnaud Perrin

    Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore
    Competing interests
    Arnaud Perrin, Patent application PCT/SG2022/050888.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3545-5470
  4. Talal Bin Amin

    Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore
    Competing interests
    No competing interests declared.
  5. Zenia Tiang

    Cardiovascular Disease Translational Research Programme, National University of Singapore, Singapore, Singapore
    Competing interests
    No competing interests declared.
  6. Dang Vinh Do

    Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore
    Competing interests
    No competing interests declared.
  7. Jiaxu Wang

    Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore
    Competing interests
    No competing interests declared.
  8. Joanna Kia Min Tan

    Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore
    Competing interests
    No competing interests declared.
  9. Shirley Suet Lee Ding

    Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore
    Competing interests
    No competing interests declared.
  10. Wei Xuan Tan

    Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore
    Competing interests
    No competing interests declared.
  11. Chang Jie Mick Lee

    Cardiovascular Disease Translational Research Programme, National University of Singapore, Singapore, Singapore
    Competing interests
    No competing interests declared.
  12. Adrian KK Teo

    Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5901-7075
  13. Roger Foo

    Cardiovascular Disease Translational Research Programme, National University of Singapore, Singapore, Singapore
    For correspondence
    roger.foo@nus.edu.sg
    Competing interests
    Roger Foo, Patent application PCT/SG2022/050888.

Funding

Biomedical Research Council (1610851033)

  • Matias I Autio

Agency for Science, Technology and Research (202D8020)

  • Matias I Autio

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Tony Yuen, Icahn School of Medicine at Mount Sinai, United States

Ethics

Animal experimentation: All animal experiments were reviewed and approved ethics and animal care committees (IRB approval: A*STAR IRB 2020-096 & IACUC: 181366 and 221660).

Version history

  1. Preprint posted: December 9, 2021 (view preprint)
  2. Received: April 21, 2022
  3. Accepted: December 28, 2023
  4. Accepted Manuscript published: January 2, 2024 (version 1)
  5. Version of Record published: February 2, 2024 (version 2)

Copyright

© 2024, Autio et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Matias I Autio
  2. Efthymios Motakis
  3. Arnaud Perrin
  4. Talal Bin Amin
  5. Zenia Tiang
  6. Dang Vinh Do
  7. Jiaxu Wang
  8. Joanna Kia Min Tan
  9. Shirley Suet Lee Ding
  10. Wei Xuan Tan
  11. Chang Jie Mick Lee
  12. Adrian KK Teo
  13. Roger Foo
(2024)
Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells
eLife 13:e79592.
https://doi.org/10.7554/eLife.79592

Share this article

https://doi.org/10.7554/eLife.79592

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