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Design Principles of a Novel Construct for HBB Gene-Editing and Investigation of Its Gene-Targeting Efficiency in HEK293 Cells

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Abstract

Beta-thalassemia is one of the most common monogenic inherited disorders worldwide caused by different mutations in the hemoglobin subunit beta (HBB) gene. Genome-editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has raised the hope for life-long gene therapy of beta-thalassemia. In a proof-of-concept study, we describe the detailed design and assess the efficacy of a novel homology-directed repair (HDR)-based CRISPR construct for targeting the HBB locus. The selected sgRNAs were designed and cloned into an optimized CRISPR plasmid. The HDR donor templates containing a reporter and a selection marker flanked by the piggyBac Inverted Tandem Repeat (ITRs), the homology arms and the delta thymidine kinase (ΔTK) gene for negative selection were constructed. The efficiency of on-target mutagenesis by the eSpCas9/sgRNAs was assessed by mismatch assays. HDR-positive cells were isolated by treatment with G418 or selection based on truncated Neuron Growth Factor Receptor (tNGFR) expression using the Magnetic Activated Cell Sorting (MACS) method followed by ganciclovir (GCV) treatment to eliminate cells with random genomic integration of the HDR donor template. In–out PCR and sanger sequencing confirmed HDR in the isolated cells. Our data showed ~ 50% efficiency for co-transfection of CRISPR/donor template plasmids in HEK293 cells and following G418 treatment, the HDR efficiency was detected at ~ 37.5%. Moreover, using a clinically-relevant strategy, HDR events were validated after selection for tNGFR+ cells followed by negative selection for ΔTK by GCV treatment. Thus, our HDR-based gene-editing strategy could efficiently target the HBB locus and enrich for HDR-positive cells.

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Data availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

This project was supported by the National Institute for Medical Research Development (NIMAD) Grant (Application Number: 973542).

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  1. Malihe Lotfi and Atefeh Ashouri have contributed equally to this work.

Authors and Affiliations

  1. Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

    Malihe Lotfi, Atefeh Ashouri, Sina Mozaffari-Jovin & Mohammad Reza Abbaszadegan

  2. Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

    Malihe Lotfi, Atefeh Ashouri, Majid Mojarrad, Sina Mozaffari-Jovin & Mohammad Reza Abbaszadegan

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Contributions

ML and AA contributed equally to this work and performed most of the experiments and wrote the manuscript with the help from other authors. MM contributed to the project design. SM-J and MRA designed and supervised the project and contributed to the interpretation of the results and manuscript writing. MRA provided the funding for the project from his grant.

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Correspondence to Sina Mozaffari-Jovin or Mohammad Reza Abbaszadegan.

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Lotfi, M., Ashouri, A., Mojarrad, M. et al. Design Principles of a Novel Construct for HBB Gene-Editing and Investigation of Its Gene-Targeting Efficiency in HEK293 Cells. Mol Biotechnol 66, 517–530 (2024). https://doi.org/10.1007/s12033-023-00739-6

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