Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells
- Agency for Science, Technology and Research, Singapore
- National University of Singapore, Singapore
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
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Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cellsDryad Digital Repository, doi:10.5061/dryad.p8cz8w9ww.
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Median gene-level TPM by tissue. Median expression was calculated from the fileGTExportal, GTEx_Analysis_2017-06-05_v8_RNASeQCv1.1.9_gene_tpm.gct.gz.
Article and author information
Author details
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
- 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
- Preprint posted: December 9, 2021 (view preprint)
- Received: April 21, 2022
- Accepted: December 28, 2023
- Accepted Manuscript published: January 2, 2024 (version 1)
- 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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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
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