Advances in Genome Editing
Yashwant Pathak in Gene Delivery, 2022
The TALEN (Transcription Activator-Like Effector Nucleases) tool was introduced in 2011 to improve the efficiency, reliability, and accessibility of genome editing. The transcription activator-like effectors (TALES) generated by the phytopathogenic bacteria Xanthomonas genus gave rise to the TALEN system (Moore et al., 2014). The activator proteins are members of the DNA binding protein family and, like transcription factors in eukaryotic genomes, can be utilized to promote the expression of their target genes. The TALE proteins’ DNA binding domain is constituted of monomers of 34 amino acid residues, each of which binds one nucleotide in the target nucleotide sequence (Christian et al., 2012). Apart from two hypervariable amino acids (the repeat variable di-residues) at positions 12 and 13, the amino acid sequence of each repeat is fundamentally similar and in charge of identifying a specific nucleotide. (Mak et al., 2013). To produce TALEN-induced targeted genomic alteration, the functional endonuclease FokI is artificially coupled to DNA binding domains to form site-specific DSBs and encourage DNA recombination. The FokI cleavage domain must be dimerized in order to cleave the two strands of the targeted DNA. As a result, TALEN modules are built in pairs to engage opposing DNA target loci, with sufficient separation (12–20 bp) between the two binding sites (Li et al., 2011).
The science of biotechnology
Ronald P. Evens in Biotechnology, 2020
In this short overview, five methods for gene editing will be defined and briefly outlined: (1) ARCUT, (2) meganucleases, (3) ZFN, (4) TALEN, and (5) CRISPER/Cas. ARCUT is artificial restriction DNA cutter. The DNA cleavage involves a pseudo-complementary peptide nucleic acid that specifies the cleavage site, DNA excision and splicing with ethylenediaminetetraacetic acid and cerium, and DNA ligase to foster DNA attachment at the target site. Meganucleases are large protein enzymes that are many in number and naturally occurring and that excise DNA sequences. They are bound to proteins that assist in specifying DNA cleavage sites. They are limited by also naturally occurring repair processes in cells that can also cause changes in other DNA sites. Zinc finger nucleases (ZFNs) are synthetic programmable combinations of a restriction endonuclease (FokI) and small zinc-ion regulated binding domain proteins, which target triple codons (three nucleic acid sites). FokI nucleases are the DNA cleavage domain only with deletion of the DNA recognition domain. FokI requires homodimerization at the target site in order to cleave DNA, such that two zinc finger molecules are needed to target two nearby DNA sites for DNA cleavage. TALEN is a transcription activator-like effector nuclease, a synthetic construction of a restriction endonuclease (FokI also), bound to a DNA-binding protein domain (TAL effector). The TALEN can bind to single nucleic acids and functions similar to the ZFNs.
Bioengineering and the Idea of Precision Medicine
Emmanuel A. Kornyo in A Guide to Bioethics, 2017
The ZFN and TALEN are nucleases that recognize DNA domains in a genome.11 The ZFN is protein-binding nuclease that breaks or cuts double-stranded DNA leading to a kind of site-specific mutagenesis. The cut leads to an activation of the DNA natural repair restriction modification mechanism (RM) to join the cuts by homologous and nonhomologous end joining (NHEJ) and this fixes the break. ZFNs could be introduced into embryonic cells by microinjection in anticipation of specific protein target for modifications such as deletions (knockout genes). Once the ZFNs reach their target and causes a break, NHEJ occurs leading to a mismatch of the DNA strand and loss of function of the genes since it has been eviscerated or spliced out.12 It is important to note that this newly modified gene is inheritable and passed onto the next generation. TALENs have almost the same mechanism as ZFN. ZFN is currently being used in clinical trial to study the mechanism of HIV infection.13 TALENs are made up of 33–34 amino acids fused with FokI nuclease to ensure specific cleavage of nucleotides.14 Typically, TALEN induces a DSB in the DNA or gene of interest and inactivate them.
The application of gene silencing in proteomics: from laboratory to clinic
Published in Expert Review of Proteomics, 2018
Maura Brioschi, Cristina Banfi
In order to obtain complete knockout of the gene of interest, TALE-DBDs have been fused with a nuclease effector domain, Folk, generating the so-called TALEN system. Folk acts only as a dimer, generated when are concomitantly present two DBDs, which are specific for two adjacent regions of the genome, and leads to a highly specific DSB that cannot be completely solved by the DNA repair system. The DSB can be repaired either by nonhomologous end joining (NHEJ), generating small unpredictable insertions or deletions (indel) responsible for impactful and inactivating mutations in the targeted sequence, or homology-directed repair (HDR), with a precise insertion of DNA at specific sites [33]. Use of TALEN pairs on the exon in 5′ of a gene induces early frameshift or stop codon, which will be permanently present in the genome, and not transient like RNAi, leading to stable knockout of the gene of interest [29].
Genome-wide CRISPR screens for the identification of therapeutic targets for cancer treatment
Published in Expert Opinion on Therapeutic Targets, 2020
Vivian Weiwen Xue, Sze Chuen Cesar Wong, William Chi Shing Cho
TALENs were discovered from Xanthomonas. Each repeat domain of TALE recognizes a single base of DNA, which is the main improvement of TALENs compared to ZFNs in genome editing. TALEN is generated by the artificial chimeric fusion of the Fok I DNA cleaving domain to TALE modules. Compared to ZFNs, TALENs are more powerful tools for generating targeted mutagenesis, but their editing efficiency will be limited by the high ratio of CpG regions in targeted sites in the genome [14]. TALENs are widely used in chimeric antigen receptor (CAR) T cell editing and related cancer immunotherapy. Sachdeva et al. applied TALEN-mediated depletion of granulocyte-macrophage colony-stimulating factor (GM-CSF) in CAR-T cells to prevent the cytokine release syndrome and subsequential secretion of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and IL-8, which aims to minimize the side effects of CART therapy in cancer treatment [15]. Besides, TALENs show potential in the clinical practice of allogeneic CAR-T cancer therapy. It was reported that TALEN-mediated editing for B cell maturation antigen receptor on T cells from healthy donors helps to avoid lymphodepletion and increased antitumor immune responses in multiple myeloma [16]. Additionally, TALEN-mediated genome editing is commonly used to engineer human stem cells for research and personalized medicine [17,18].
Advances on potential therapeutic options for epidermolysis bullosa
Published in Expert Opinion on Orphan Drugs, 2018
Laura De Rosa, Ulrich Koller, Johann W. Bauer, Michele De Luca, Julia Reichelt
Gene editing tools have evolved at a tearing pace over the past few years starting with meganucleases and zinc-finger nucleases and moving rapidly on to transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). The simplicity of designing TALEN and even more so CRISPR led to a much wider use and further development of the latter. Although both TALEN and CRISPR consist of a DNA binding domain and a nuclease domain, the binding chemistry of the two entities to DNA target sequences is entirely distinct from each other, with consequences for both targeting efficiency and specificity (for review see [29]).
Related Knowledge Centers
- Crispr
- Genome
- Nuclease
- Restriction Enzyme
- Xanthomonas
- Transcription Activator-Like Effector
- DNA-Binding Domain
- In Situ
- Zinc-Finger Nuclease
- Type III Secretion System