The process and implications of gene editing in humans

By Pia Skok

            Gene editing is a process during which nuclease-based gene editing machinery is delivered into the cell where it adds and deletes genes as well as performs other highly targeted site-specific genomic modifications using DNA repair mechanisms.¹ This allows us to create precisely manipulated genomes of cells or organisms to gain or modify a specific characteristic.² Such changes to the genetic constitution of a living cell have implications in many areas including basic and biomedical research as well as in medicine for treatment of diseases. 

            The process of gene editing begins by delivering the gene editing machinery to the cell. This can be done via formation of a recombinant plasmid, viral vector or lipid nanoparticle which carry the genes encoding the editing machinery. In the cell, the newly introduced DNA is translated, and the gene editing machinery is synthesised.³ The gene editing machinery is based on highly specific and programmable nucleases which induce double-stranded breaks (DSBs) at a specific site in the genome. These breaks lead to stimulation of cell’s own repair mechanisms including homology directed repair and non-homologous end joining.⁴ The outcome of these processes depends on the number of DSBs introduced as well as presence of exogenous DNA that acts as template. Non-homologous end joining can induce unpredictable insertions, deletions, inversion, and duplications, while homology-directed repair can induce precise base mutations, insertions, deletions, and replacement in the targeted area.⁵

The three most used nucleases currently available are: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein (CRISPR/Cas) system. All of them follow a similar general mechanism of introducing site-specific DSBs followed by cell’s repair mechanisms, but also have their own specific characteristics that must be considered when selecting the most suitable one for a specific application.² For example, since ZFNs are guided by zinc-finger domains their specificity does not depend on the target sequence to be cleaved, but rather on adjacent sequences in the genome. This can lead to non-specific off-target cleavages which causes genome fragmentation and instability as well as toxicity to cells. TALENs introduce more specific and efficient DSBs however, their size impedes the delivery to the cell. CRISPR/Cas system is RNA based which allows modification of several genomic sites simultaneously as well as easy design for any genomic target as only guide RNA needs to be changed while the nuclease remains the same so no need for complicated manipulations in the protein domain.²

The ability of the abovementioned gene editing machinery to target and manipulate the genome of cells and organisms has a wide range of implications in basic and biomedical research as well as in medicine. 

Gene editing can be used in basic research to study gene function and regulation. CRISPR-Cas gene editing machinery has revolutionized our ability to study gene function by introducing loss-of-function mutations or causing gene deletions. For example, CRISPR/Cas9 has been used to study the function of various protein coding genes involved in cytokinesis. By introducing loss-of-function mutations it was shown that chloride intracellular channel 4 regulates the cortical actin network and without it cells fail to undergo cytokinesis.⁶ Studying gene regulation is even more challenging than studying its function due to the numerous and widespread regulatory regions that interact with each other to control gene expression. To provide insights into how multiple regulatory elements interact and collectively contribute to the expression and switching of fetal haemoglobin, CRISPR/Cas9 system was used to make multiple specific mutations in its regulatory regions. Prior to gene editing technology, mechanisms of fetal haemoglobin regulation were elucidated through studies of human genetic variation, which lead to substantial insights but failed to reveal specific genetic changes and how they act individually and in combination to regulate expression.⁷

In addition, to elucidating protein function and regulation, gene editing technology plays a vital role in biomedical research. By creating new model organisms and cell-based disease models, it improves our understanding of genetic mechanisms governing the development of diseases. TALENs have been used to inactivate the LDL receptor gene in pigs, thus generating a model for familial hypercholesterolemia. This model can be used to learn more about the condition as well as for treatment development.⁸ Furthermore, CRISPR/Cas9 has been used to achieve site-specific editing of non-coding genomic regions in native human islets to obtain a more accurate cell-based model that allows us to perform functional studies of non-coding genomic regions leading to a major advance for islet biology and diabetes research.⁹

Gene editing also offers advances in the medical field.  By correcting or disrupting the sequences of genes that cause the disease, it can be used as a potential alternative to therapies that only treat symptoms.⁸ Gene editing can be applied both in vivo and ex vivo. Ex vivo gene editing has been used in AIDS treatment. ZFNs modify infection-related genes to produce HIV-resistant CD4+ T cells which are subsequently reinfused into patients. In contrast, transthyretin amyloidosis, disease characterized by progressive accumulation of misfolded transthyretin, can be treated with in vivo gene editing. CRISPR-Cas9 is delivered to hepatocytes where it disables the transthyretin gene. Although both the diseased and the healthy allele are disrupted in the process, this does not have major pathological consequences, as transthyretin is only involved in transport of retinoic acid, the loss of which can be overcome by vitamin A supplementation. CRISPR/Cas9 thus provides a very efficient way to treat transthyretin amyloidosis.¹

In conclusion, nuclease-based gene editing machinery that triggers cell’s repair mechanisms to cause site-specific genomic modifications, has many research and therapeutic implications.  Despite the positive outcomes already attained, the genome editing techniques still have some limitations. For example, temporal and spatial control of editing machinery’s expression, its delivery and targetability as well as its accuracy.¹⁰ These important challenges remain to be addressed in order to take advantage of gene editing’s full potential and use it routinely for research and therapeutic applications.

References:

1. Li, H. et al. 2020. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Sig Transduct Target Ther 5(1). doi:  https://doi.org/10.1038/s41392-019-0089-y
2. Khalil, A.M. 2020. The genome editing revolution: review. J Genet Eng Biotechnol 18(68). doi: https://doi.org/10.1186/s43141-020-00078-y
3. Ahmar, S. et al. 2020. A Revolution toward Gene-Editing Technology and Its Application to Crop Improvement. International Journal of Molecular Sciences 21(16): 5665. doi: 10.3390/ijms21165665
4. Rodríguez‑Rodríguez, D.R. et al. 2019. Genome editing: A perspective on the application of CRISPR/Cas9 to study human diseases (Review). International Journal of Molecular Medicine 43: 1559-1574. doi: https://doi.org/10.3892/ijmm.2019.4112
5. Yue, M. et al. 2016. Recent progress in CRISPR/Cas9 technology. Journal of Geetics and Genomics 43(2). doi: 10.1016/j.jgg.2016.01.001
6. Husser, M. C. et al. 2021. CRISPR-Cas tools to study gene function in cytokinesis. J Cell Sci 134 (8). doi: https://doi.org/10.1242/jcs.254409
7. Shen, Y. et al. 2021. A unified model of human hemoglobin switching through single-cell genome editing. Nat Commun 12, 4991. doi: https://doi.org/10.1038/s41467-021-25298-9
8. Joung, J. and Sander, J. 2013. TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 14,49–55. doi: https://doi.org/10.1038/nrm3486
9. Bevacqua, R. J. et al. 2021. CRISPR-based genome editing in primary human pancreatic islet cells. Nat Commun 12,2397. oi: https://doi.org/10.1038/s41467-021-22651-w

10. Lubroth, P. et al. 2021. In vivo Genome Editing Therapeutic Approaches for Neurological Disorders: Where Are We in the Translational Pipeline? Front. Neurosci. doi: https://doi.org/10.3389/fnins.2021.632522

Article written in June, 2022