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Understanding the Technologies Involved in Gene Therapy
Published in Yashwant V. Pathak, Gene Delivery Systems, 2022
Manish P. Patel, Jayvadan K. Patel, Mukesh Patel, Govind Vyas
With the help of ZFNs, the gene of interest can either be knocked in or knocked out. Regardless of the goal, two things are required: the cell line of interest where gene editing is to be performed and a plasmid that contains a nuclease sequence. Once the cell line and integration-defective lentiviral vectors or plasmid encoded with a sequence of nuclease proteins are obtained, the cell needs to be transfected with the plasmid or integration-defective lentiviral vector. This sequence transcribes to form messenger RNA (mRNA) and will be translated then to produce ZFN proteins in the cell. This protein enters the nuclease and binds with the specific target sequence with the help of the DNA binding domain part of the nuclease. After that, it cleaves the specific gene of interest in DNA with the help of the FokI catalytic domain. FokI are only active in a dimerized conformation. Thus, to achieve functionality, two identical domains need to dimerize around one target DNA (Akçay et al. 2014). This cleavage produces a double-strand break (DSB) in DNA. This DSB repair occurs by two pathways in eukaryotic cells: homologous recombination (HR) and non-homologous end joining (NHEJ). During NHEJ repair, the two broken ends of DNA are rapidly and efficiently ligated together, frequently with the introduction of small insertions or deletions at the region, which can lead to gene disruption (Jo et al. 2015).
Application of CRISPR-Cas Genome Editing Tools for the Improvement of Plant Abiotic Stress Tolerance
Published in Hasanuzzaman Mirza, Nahar Kamrun, Fujita Masayuki, Oku Hirosuke, Tofazzal M. Islam, Approaches for Enhancing Abiotic Stress Tolerance in Plants, 2019
Pankaj Bhowmik, Md. Mahmudul Hassan, Kutubuddin Molla, Mahfuzur Rahman, M. Tofazzal Islam
The DSBs introduced by the CRISPR-Cas9 complex can be repaired by non-homologous end joining (NHEJ) and homologous recombination (HR) (Figure 26.2A). The homologous end joining or recombination is also popularly known as Homology Dependent Repair (HDR). The NHEJ repair can produce two different mutations at each chromosome: heterozygous mutations, biallelic mutations, and two independent identical mutations: homozygous mutations leading to gene insertion or gene deletion (Figure 26.2). In the presence of donor DNA digested with the same endonuclease leaving behind similar overhangs, HEJ can be achieved, leading to gene modification and insertion.
Genome Editing for Genetic Lung Diseases
Published in Anthony J. Hickey, Sandro R.P. da Rocha, Pharmaceutical Inhalation Aerosol Technology, 2019
Genome editing is a type of DNA engineering which allows precise modifications in the genomic DNA. Genome editing is mediated through programmable nucleases which recognize and bind specific sequences in the genome.1,2 Once binding, the nucleases can be engineered to cut targeted genomic DNA, resulting in double-strand breaks (DSBs), which are efficiently repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) in the presence of a donor DNA template.1–5 NHEJ is error-prone, often introducing small or large insertions or deletions (indels) at the DNA cut site.6 Depending on the types and positions, indels that shift the open reading frame (ORF) can lead to the mRNA degradation or production of abnormal and non-functional proteins.7 Genome editing nucleases-mediated NHEJ is able to introduce long-term disruption of disease-prone genes.8 It is also feasible to engineer nucleases-mediated NHEJ to restore ORF of a malfunctioning gene.9–13 For example, mutations in dystrophin gene can lead to ORF shifting and severe disruption or deletion of dystrophin protein, to cause Duchenne muscular dystrophy. Disruption of the ORF-shifted exons to restore the ORF can rescue the function of dystrophin for treating Duchenne muscular dystrophy.9–13 In contrast to error-prone NHEJ pathway, HDR-mediated genome editing enables precise modifications by incorporation of the donor DNA with homologous sequences.14 Genome editing via HDR pathway could be used to precisely repair mutations or knock-in sequences at the desired loci.15–17 Besides introducing DSBs, genome editing nucleases (e.g. Cas9) can be engineered to enable many different modifications, including epigenetic modification of targeted sequences, induction or suppression of gene expression, base editing, imaging of genomic locus, and many others.8,18–25
The potential for the use of gene drives for pest control in New Zealand: a perspective
Published in Journal of the Royal Society of New Zealand, 2018
Peter K. Dearden, Neil J. Gemmell, Ocean R. Mercier, Philip J. Lester, Maxwell J. Scott, Richard D. Newcomb, Thomas R. Buckley, Jeanne M. E. Jacobs, Stephen G. Goldson, David R. Penman
This technology requires, however, the target site to be invariant, to match the gRNA in the species being targeted. Variations in the target site could occur in the population naturally, or could be caused by the gene drive mechanism itself. In the example shown in Figure 1, HDR of the Cas9-induced double-stranded break is critical for homing. Most cells also have an alternative pathway for repairing double-stranded breaks, known as non-homologous end joining (NHEJ) (Carroll 2014). With NHEJ, the broken ends are just ligated together, often causing errors such as deletions or insertions. A NHEJ-mediated mutation of the Cas9 recognition site would suppress homing (Burt 2003), meaning that spread of the gene drive system would be blocked and pest suppression would fail.