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Combination of a TALEN pair into one plasmid vector. A. Left and right TALENs were ligated into different Entry plasmids to produce the “2 in 1” TALEN system. The left TALEN Entry vector contained attL1 and attR5 flanking sequences and the EGFP reporter. The right TALEN Entry vector contained attL5 and attL2 flanking sequences and the mCherry reporter. The left and right TALEN Entry vectors were then combined into the pPB-DEST destination vector by Gateway® LR recombination. In some cases Pgk-Puro was added to the destination vector to facilitate selection. B-D Example of transfection of Ddx3x TALEN plasmids into mouse Neuro-2a cells. B1 and B2: left Ddx3x TALEN Entry plasmid. C1 and C2, right Ddx3x TALEN Entry plasmid. D1 to D4, “2 in 1” Ddx3x TALEN plasmid. B1, C1, D1, phase contrast; B2, C2, D2, D3, D4, epifluorescence. E. Surveyor mutation analysis of the Ddx3x gene after transfection of the individual left (lane 1) or right (lane 2) Ddx3x TALEN Entry plasmids or the “2 in 1“ Ddx3x TALEN plasmid (lane 3) into Neuro-2a cells.
Source publication
Transcriptional activator-like effector nucleases (TALENs) have become a powerful tool for genome editing. Here we present an efficient TALEN assembly approach in which TALENs are assembled by direct Golden Gate ligation into Gateway(®) Entry vectors from a repeat variable di-residue (RVD) plasmid array. We constructed TALEN pairs targeted to mouse...
Contexts in source publication
Context 1
... assembly of RVDs into these Entry plasmids followed the same procedure described above. We also constructed two PiggyBac transposon-based destination vectors, pPB-DEST and pPB- DEST-Puro ( Figure 4A). This system can be readily modified for other destination vector systems. ...
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... should be pointed out that it is impossible to sequence the RVDs in the final vectors. Nonetheless, we transfected each TALEN Entry plasmid ( Figure 4B and C) and the TALEN "2 in 1" plasmid ( Figure 4D) into Neuro-2a cells. Surveyor ® mutation analysis demonstrated that the TALEN pair assembled in one plasmid ("2 in 1") was capable of introducing mutations in genome ( Figure 4E). ...
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... should be pointed out that it is impossible to sequence the RVDs in the final vectors. Nonetheless, we transfected each TALEN Entry plasmid ( Figure 4B and C) and the TALEN "2 in 1" plasmid ( Figure 4D) into Neuro-2a cells. Surveyor ® mutation analysis demonstrated that the TALEN pair assembled in one plasmid ("2 in 1") was capable of introducing mutations in genome ( Figure 4E). ...
Similar publications
In the recent years, sequence-specific nucleases such as ZFNs, TALENs, and CRISPR/Cas9 have revolutionzed the fields of animal genome editing and transgenesis. However, these new techniques require microinjection to deliver nucleic acids into embryos to generate gene-modified animals. Microinjection is a delicate procedure that requires sophisticat...
Citations
... Similarly, TALEN methods have undergone significant improvements in design, assembly, and delivery, resulting in improved precision of genetic modifications (Sakuma et al., 2013). Emerging technology platforms now facilitate automated, high-throughput assembly of customized TALENs (Zhang et al., 2013). In addition, the combined use of TALENs and CRISPR technologies promotes a synergistic effect that optimizes precision and streamlines the editing process (Xu et al., 2015). ...
Pediatric cancer continues to be a global health issue needing the pursuit of innovative treatment options. One emerging area is gene therapy, holding the potential for dramatic improvements in pediatric cancer care. This review comprehensively overviews recent developments and challenges in pediatric cancer gene therapy. It starts by explaining the concepts underlying gene therapy, including gene delivery systems, gene editing techniques, and the logic of targeted therapies. It then goes through recent advances, highlighting meaningful progress in using both viral and non-viral vectors to deliver genes to cancer cells and specifically effectively, it also examines the development and use of gene-editing tools like CRISPR/Cas9, being used to modify cancer-linked genes and enhance treatment results. This review offers a complete look at the different gene treatments used for cancers in kids. That includes cell therapies, viruses that target cancer cells, and gene tweaks that change the immune response. It breaks down how they work, what clinical trials show and where they fall short. The authors really dive into the challenges of taking these therapies from animal studies to actual patients - things like making good lab models and getting government approval. Most importantly, the review talks about the huge strides made recently in gene therapy for young cancer patients. but it also admits there's still a long way to go in making treatments better and improving quality of life. and Mapping out the landscape like this hopefully moves things along so more children can survive cancer.
... TALENs are transcription activator-like effector nucleases developed from effector TALEs in Xanthomonas [109]. TALENs are modular in form and function like ZFNs, consisting of a fusion of the FokI cleavage domain and TALE protein DNA-binding domain [19]. ...
Gene editing (GE) has become one of the mainstream bioengineering technologies over the past two decades, mainly fueled by the rapid development of the CRISPR/Cas system since 2012. To date, plenty of articles related to the progress and applications of GE have been published globally, but the objective, quantitative and comprehensive investigations of them are relatively few. Here, 13,980 research articles and reviews published since 1999 were collected by using GE-related queries in the Web of Science. We used bibliometric analysis to investigate the competitiveness and cooperation of leading countries, influential affiliations, and prolific authors. Text clustering methods were used to assess technical trends and research hotspots dynamically. The global application status and regulatory framework were also summarized. This analysis illustrates the bottleneck of the GE innovation and provides insights into the future trajectory of development and application of the technology in various fields, which will be helpful for the popularization of gene editing technology.
... TALENs, are isolated from the pathogenic bacteria Xanthomonas [368]. TALENs have remarkable advantages compared to ZFNs, such as lower cytotoxicity [369] and higher targeting range [370]. However, there are no single site-specific TALENs with long arrays due to the lack of the linkage between repeats. ...
Regulating cellular uptake pathways using engineered materials is becoming a vital strategy for efficient gene transfer because the success of gene delivery most often relies on the uptake mechanism and the intracellular fate of the delivery vectors. The uptake of gene carriers can be greatly affected by the various physical, geometrical, chemical, and biological characteristics of the delivery vectors. In the design of gene delivery materials, especially polymeric materials, it is important to understand not only how gene carriers are taken up and transported into cells, but also how the uptake mechanism can be regulated. In this review, we discuss polymeric materials that regulate cellular uptake pathways for highly effective delivery of gene therapeutics, elucidate various routes of cellular uptake that alter the intracellular fate of polymeric gene carriers and finding efficient strategies for overcoming extracellular and intracellular obstacles. We also discuss the structures of polymeric materials in order to understand how they regulate cellular uptake. Lastly, we discuss various strategic approaches, including essential cues on how to regulate the cellular uptake pathways of polymeric carriers and how to control their endocytic trafficking to improve the efficacy of gene delivery.
... TALEN nucleases were first discovered in the pathogenic bacterium Xanthomonas [35]. TALE proteins contain about 30 amino acid repeats flanking the DNA-binding region. ...
CRISPR-Cas systems, widespread in bacteria and archaea, are mainly responsible for adaptive cellular immunity against exogenous DNA (plasmid and phage). However, the latest research shows their involvement in other functions, such as gene expression regulation, DNA repair and virulence. In recent years, they have undergone intensive research as convenient tools for genomic editing, with Cas9 being the most commonly used nuclease. Gene editing may be of interest in biotechnology, medicine (treatment of inherited disorders, cancer, etc.), and in the development of model systems for various genetic diseases. The dCas9 system, based on a modified Cas9 devoid of nuclease activity, called CRISPRi, is widely used to control gene expression in bacteria for new drug biotargets validation and is also promising for therapy of genetic diseases. In addition to direct use for genomic editing in medicine, CRISPR-Cas can also be used in diagnostics, for microorganisms' genotyping, controlling the spread of drug resistance, or even directly as "smart" antibiotics. This review focuses on the main applications of CRISPR-Cas in medicine, and challenges and perspectives of these approaches.
... For instance, ZFNs were delivered by adenoviral vectors. 7,[11][12][13] Similarly, intact TALEN sequences could be maintained as individual expression cassettes in firstand second-generation adenoviral vectors, [14][15][16] as well as a complete TALEN pair in HCAdV. 7,17 The CRISPR/Cas9 system was packed in early generation adenoviral vectors, too, including the expression cassettes of Cas9 and guide RNA (gRNA) apart 18 or in combination. ...
High-capacity adenoviral vectors (HCAdVs) devoid of all
coding genes are powerful tools to deliver large DNA
cargos into cells. Here HCAdVs were designed to deliver a
multiplexed complete CRISPR/Cas9 nuclease system or a
complete pair of transcription activator-like effector nucleases
(TALENs) directed against the hepatitis B virus (HBV)
genome. HBV, which remains a serious global health burden,
forms covalently closed circular DNA (cccDNA) as a persistent
DNA species in infected cells. This cccDNA promotes
the chronic carrier status, and it represents a major hurdle
in the treatment of chronic HBV infection. To date, only
one study demonstrated viral delivery of a CRISPR/Cas9
system and a single guide RNA (gRNA) directed against
HBV by adeno-associated viral (AAV) vectors. The advancement
of this study is the co-delivery of multiple gRNA
expression cassettes along with the Cas9 expression cassette
in one HCAdV. Treatment of HBV infection models resulted
in a significant reduction of HBV antigen production and
the introduction of mutations into the HBV genome. In
the transduction experiments, the HBV genome, including
the HBV cccDNA, was degraded by the CRISPR/Cas9 system.
In contrast, the combination of two parts of a TALEN pair in
one vector could not be proven to yield an active system. In
conclusion, we successfully delivered the CRISPR/Cas9 system
containing three gRNAs using HCAdV, and we demonstrated
its antiviral effect.
... For instance, ZFNs were delivered by adenoviral vectors. 7,[11][12][13] Similarly, intact TALEN sequences could be maintained as individual expression cassettes in firstand second-generation adenoviral vectors, [14][15][16] as well as a complete TALEN pair in HCAdV. 7,17 The CRISPR/Cas9 system was packed in early generation adenoviral vectors, too, including the expression cassettes of Cas9 and guide RNA (gRNA) apart 18 or in combination. ...
... Four years following the development of ZFNs, a new class of natural DNA-binding proteins was discovered in the plant pathogenic bacteria Xanthomonas sp. ( Zhang et al., 2013). The proteins, termed transcription activator-like effectors (TALEs), contain 33-35 amino acid repeats that flank a central DNA binding region (amino acids 12 and 13). ...
Gene therapy has long held promise to correct a variety of human diseases and defects. Discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), the mechanism of the CRISPR-based prokaryotic adaptive immune system (CRISPR-associated system, Cas), and its repurposing into a potent gene editing tool has revolutionized the field of molecular biology and generated excitement for new and improved gene therapies. Additionally, the simplicity and flexibility of the CRISPR/Cas9 site-specific nuclease system has led to its widespread use in many biological research areas including development of model cell lines, discovering mechanisms of disease, identifying disease targets, development of transgene animals and plants, and transcriptional modulation. In this review, we present the brief history and basic mechanisms of the CRISPR/Cas9 system and its predecessors (ZFNs and TALENs), lessons learned from past human gene therapy efforts, and recent modifications of CRISPR/Cas9 to provide functions beyond gene editing. We introduce several factors that influence CRISPR/Cas9 efficacy which must be addressed before effective in vivo human gene therapy can be realized. The focus then turns to the most difficult barrier to potential in vivo use of CRISPR/Cas9, delivery. We detail the various cargos and delivery vehicles reported for CRISPR/Cas9, including physical delivery methods (e.g. microinjection; electroporation), viral delivery methods (e.g. adeno-associated virus (AAV); full-sized adenovirus and lentivirus), and non-viral delivery methods (e.g. liposomes; polyplexes; gold particles), and discuss their relative merits. We also examine several technologies that, while not currently reported for CRISPR/Cas9 delivery, appear to have promise in this field. The therapeutic potential of CRISPR/Cas9 is vast and will only increase as the technology and its delivery improves.
... The purpose of targeting host proteins is to produce a functional cure, thereby suppressing viral replication and restoring a functional immune system in the presence of infection. Thus far, the objective of targeting HIV genes is to damage the proviral DNA in infected Table 1 Characteristics of viral vectors targeting T cells, macrophages, dendritic cells, and hematopoietic stem cells for HIV-1 gene therapy Table adapted from (Schroers et al. 2004;Zhang et al. 2013;Ayuso 2016;Summerford and Samulski 1998;Finkelshtein et al. 2013 cells, thereby eradicating the latent viral reservoir. One distinct difference in these strategies is that both copies of the host genes would need to be inactivated in the diploid genome, whereas targeting HIV genes needs to inactivate the one integrated copy of HIV provirus (Josefsson et al. 2011). ...
... The viral DNA does not integrate into the host genome and transiently expresses transgenes. Infection with a set of recombinant Ad5 adenoviral vector delivered monocistronic TALEN pairs, producing editing of the Ddx3y gene (Zhang et al. 2013). Optimally, it would be better to use a bicistronic vector with the Ad5, but packaging both large TALEN genes may prove difficult. ...
Transcription activator-like effector nucleases (TALENs) are one of several types of programmable, engineered nucleases that bind and cleave specific DNA sequences. Cellular machinery repairs the cleaved DNA by introducing indels. In this review, we emphasize the potential, explore progress, and identify challenges in using TALENs as a therapeutic tool to treat HIV infection. TALENs have less off-target editing and can be more effective at tolerating HIV escape mutations than CRISPR/Cas-9. Scientists have explored TALEN-mediated editing of host genes such as viral entry receptors (CCR5 and CXCR4) and a protein involved in proviral integration (LEDGF/p75). Viral targets include the proviral DNA, particularly focused on the long terminal repeats. Major challenges with translating gene therapy from bench to bedside are improving cleavage efficiency and delivery, while minimizing off-target editing, cytotoxicity, and immunogenicity. However, rapid improvements in TALEN technology are enhancing cleavage efficiency and specificity. Therapeutic testing in animal models of HIV infection will help determine whether TALENs are a viable HIV treatment therapy. TALENs or other engineered nucleases could shift the therapeutic paradigm from life-long antiretroviral therapy toward eradication of HIV infection.
... Zinc finger nucleases (ZFNs) are the first sequence-specific designer nucleases for gene editing. ZFNs consist of a DNA recognition domain (Zinc finger proteins, ZFPs) which is composed of 3-6 Cys 2 -His 2 zinc fingers and a non-specific DNA cleavage domain (derived from FokI endonucleases) via the C-terminal of the cleavage domain [17,18]. Each zinc finger domain of ZFPs can recognize 3-to 4-bp DNA arrays via a single α-helix, and several tandem zinc finger domains can typically recognize and bind to DNA sequences with the length of multiple of three, normally 9-to 18-bp with high specificity [19]. ...
... The DNA-binding domain is composed of multiple units of 33~35 amino acid repeat arrays arranged in tandem, and it is contained in TALEs, which are natural proteins of the plant pathogenic bacteria Xanthomonas sp. [18]. In contrast to ZFPs, TALENs possess advanced advantages such as lower cytotoxicity [56], greater design flexibility leading to higher targeting range [57], and easier engineering. ...
Therapeutic genome editing technology has been widely used as a powerful tool for directly correcting genetic mutations in target pathological tissues and cells to cure of diseases. The modification of specific genomic sequences can be achieved by utilizing programmable nucleases, such as Meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly-interspaced short palindromic repeat-associated nuclease Cas9 (CRISPR/Cas9). However, given the properties, such as large size, negative charge, low membrane penetrating ability, as well as weak tolerance for serum, and low endosomal escape, of these nucleases genome editing cannot be successfully applied unless in vivo delivery of related programmable nucleases into target organisms or cells is achieved. Here, we look back at delivery strategies having been used in the in vivo delivery of three main genome editing nucleases, followed by methodologies currently undergoing testing in clinical trials, and potential delivery strategies provided by analyzing characteristics of nucleases and commonly used vectors.
... To further increase the utility of the system, Golden Gate modular assembly systems were deployed using a system of unique restriction nucleases to create TALENs in two steps (26). Golden Gate assembly has since been expanded and simplified ((27, 28)) for high-throughput assembly (29), making multi-gene targeted knockout projects considerably more feasible and affordable, even for smaller laboratories. Furthermore, the TALEN system has been optimized for rapid, specific cutting that is efficiently transmitted to the germline (7,30). ...
Over the last few years, the technology to create targeted knockout and knockin zebrafish animals has exploded. We have gained the ability to create targeted knockouts through the use of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR associated system (CRISPR/Cas). Furthermore, using the high-efficiency TALEN system, we were able to create knockin zebrafish using a single-stranded DNA (ssDNA) protocol described here. Through the use of these technologies, the zebrafish has become a valuable vertebrate model and an excellent bridge between the invertebrate and mammalian model systems for the study of human disease.
