Transcription activator-like effector nuclease-mediated deletion safely eliminates the major egg allergen ovomucoid in chickens.

Among the major egg allergens, ovomucoid (OVM) is very stable against heat and digestive enzymes, making it difficult to remove physiochemically and inactivate allergens. However, recent genome editing technology has made it possible to generate OVM-knockout chicken eggs. To use this OVM-knockout chicken egg as food, it is important to evaluate its safety as food. Therefore, in this study, we examined the presence or absence of mutant protein expression, vector sequence insertion, and off-target effects in chickens knocked out with OVM by platinum TALENs. The eggs laid by homozygous OVM-knockout hens showed no evident abnormalities, and immunoblotting showed that the albumen contained neither the mature OVM nor the OVM truncated variant. Whole genome sequencing (WGS) revealed that the potential TALEN-induced off-target effects in OVM-knockout chickens were localized in the intergenic and intron regions. The WGS information confirmed that plasmid vectors used for genome editing were only transiently present and did not integrate into the genome of edited chickens. These results indicate the importance of safety evaluation and reveal that the eggs laid by this OVM knockout chicken solve the allergy problem in food and vaccines.

A Transcriptomic Analysis of Tobacco Leaf with the Functional Loss of the Plastid rpoB Operon Caused by TALEN-Mediated Double-Strand Breakage.

At least two sets of RNA polymerase (RNAP), nucleus (NEP)- and plastid (PEP)-encoded polymerases, recognizing distinct promoters exist in the plastids of land plants. Most plastid genes are regulated by multiple promoters with different strengths in their response to developmental stages and environmental cues. Recently, we applied chloroplast-targeted transcription activator-like effector nuclease (cpTALEN) technology to site-specifically cause double-strand DNA breaks in the rpoB gene of tobacco, which encodes the ß-subunit of PEP. The repair of damaged chloroplast DNA (cpDNA) through microhomology-mediated recombination caused the functional loss of the rpoB operon and resulted in the heterotrophic growth of an albino plant. We conducted a genome-wide analysis of the steady state of gene expression in the leaf tissue of PEP-deficient tobacco by RNA-Seq and compared it with that of wild-type plants. The expression of NEP genes was up-regulated in PEP-deficient tobacco; in particular, the level of RpoT3 transcripts encoding the specifically plastid-targeted NEP was significantly increased. Alongside most housekeeping genes, NEP also plays an important role in the regulation of gene expression involved in photosynthesis. In contrast, alongside the photosynthesis-related genes, PEP also plays an important role in the regulation of gene expression involved in some housekeeping functions. Furthermore, the mitochondrial DNA copy number and the level of most mitochondrial protein-coding transcripts were slightly increased in PEP-deficient tobacco. The disruption of PEP function not only affected plastid gene expression, but also nuclear and mitochondrial gene expression. This study demonstrated the intercompartmental retrograde signaling in the regulation of gene expression.

Circadian Clock Genes Regulate Temperature-Dependent Diapause Induction in Silkworm Bombyx mori.

The bivoltine strain of the domestic silkworm, Bombyx mori, exhibits a facultative diapause phenotype that is determined by maternal environmental conditions during embryonic and larval development. Although a recent study implicated a circadian clock gene period (per) in circadian rhythms and photoperiod-induced diapause, the roles of other core feedback loop genes, including timeless (tim), Clock (Clk), cycle (cyc), and cryptochrome2 (cry2), have to be clarified yet. Therefore, the aim of this study was to elucidate the roles of circadian clock genes in temperature-dependent diapause induction. To achieve this, per, tim, Clk, cyc, and cry2 knockout (KO) mutants were generated, and the percentages of diapause and non-diapause eggs were determined. The results show that per, tim, Clk, cyc, and cry2 regulated temperature-induced diapause by acting upstream of cerebral γ-aminobutyric acid (GABA)ergic and diapause hormone signaling pathways. Moreover, the temporal expression of the clock genes in wild-type (wt) silkworms was significantly different from that of thermosensitive transient receptor potential ankyrin 1 (TRPA1) KO mutants during embryonic development. Overall, the findings of this study provide target genes for regulating temperature-dependent diapause induction in silkworms.

Repairing TALEN-mediated double-strand break by microhomology-mediated recombination in tobacco plastids generates abundant subgenomic DNA.

Transcription activator-like effector nuclease (TALEN) technology has been widely used to edit nuclear genomes in plants but rarely for editing organellar genomes. In addition, ciprofloxacin, commonly used to cause the double-strand break of organellar DNA for studying the repair mechanism in plants, confers no organellar selectivity and site-specificity. To demonstrate the feasibility of TALEN-mediated chloroplast DNA editing and to use it for studying the repair mechanism in plastids, we developed a TALEN-mediated editing technology fused with chloroplast transit peptide (cpTALEN) to site-specifically edit the rpoB gene via Agrobacteria-mediated transformation of tobacco leaf. Transgenic plants showed various degrees of chlorotic phenotype. Repairing damaged plastid DNA resulted in point mutation, large deletion and small inversion surrounding the rpoB gene by homologous recombination and/or microhomology-mediated recombination. In an albino line, microhomology-mediated recombination via a pair of 12-bp direct repeats between rpoC2 and ycf2 genes generated the chimeric ycf2-rpoC2 subgenome, with the level about 3- to 5-fold higher for subgenomic DNA than ycf2. Additionally, the expression of chimeric ycf2-rpoC2 transcripts versus ycf2 mRNA agreed well with the level of corresponding DNA. The ycf2-rpoC2 subgenomic DNA might independently and preferentially replicate in plastids.

Current status of the application of gene editing in pigs.

Genetically modified animals, especially rodents, are widely used in biomedical research. However, non-rodent models are required for efficient translational medicine and preclinical studies. Owing to the similarity in the physiological traits of pigs and humans, genetically modified pigs may be a valuable resource for biomedical research. Somatic cell nuclear transfer (SCNT) using genetically modified somatic cells has been the primary method for the generation of genetically modified pigs. However, site-specific gene modification in porcine cells is inefficient and requires laborious and time-consuming processes. Recent improvements in gene-editing systems, such as zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas) system, represent major advances. The efficient introduction of site-specific modifications into cells via gene editors dramatically reduces the effort and time required to generate genetically modified pigs. Furthermore, gene editors enable direct gene modification during embryogenesis, bypassing the SCNT procedure. The application of gene editors has progressively expanded, and a range of strategies is now available for porcine gene engineering. This review provides an overview of approaches for the generation of genetically modified pigs using gene editors, and highlights the current trends, as well as the limitations, of gene editing in pigs.

A Targeted In-Fusion Expression System for Recombinant Protein Production in Bombyx mori.

The domesticated silkworm, Bombyx mori, is an economically important insect that synthesizes large amounts of silk proteins in its silk gland to make cocoons. In recent years, germline transformation strategies advanced the bioengineering of the silk gland as an ideal bioreactor for mass production of recombinant proteins. However, the yield of exogenous proteins varied largely due to the random insertion and gene drift caused by canonical transposon-based transformation, calling for site-specific and stable expression systems. In the current study, we established a targeted in-fusion expression system by using the transcription activator-like effector nuclease (TALEN)-mediated targeted insertion to target genomic locus of sericin, one of the major silk proteins. We successfully generated chimeric Sericin1-EGFP (Ser-2A-EGFP) transformant, producing up to 3.1% (w/w) of EGFP protein in the cocoon shell. With this strategy, we further expressed the medically important human epidermal growth factor (hEGF) and the protein yield in both middle silk glands, and cocoon shells reached to more than 15-fold higher than the canonical piggyBac-based transgenesis. This natural Sericin1 expression system provides a new strategy for producing recombinant proteins by using the silkworm silk gland as the bioreactor.

Preclinical Evaluation of a Novel TALEN Targeting CCR5 Confirms Efficacy and Safety in Conferring Resistance to HIV-1 Infection.

Therapies to treat patients infected with human immunodeficiency virus (HIV) aim at preventing viral replication but fail to eliminate the virus. Although transplantation of allogeneic CCR5Δ32 homozygous stem cell grafts provided a cure for a few patients, this approach is not considered a general therapeutic strategy because of potential side effects. Conversely, gene editing to disrupt the C-C chemokine receptor type 5 (CCR5) locus, which encodes the major HIV coreceptor, has shown to confer resistance to CCR5-tropic HIV strains. Here, an engineered transcription activator-like effector nuclease (TALEN) that enables efficient CCR5 editing in hematopoietic cells is presented. After transferring TALEN-encoding mRNA into primary CD4+ T cells, up to 89% of CCR5 alleles are disrupted. Genotyping confirms the genetic stability of the CCR5-edited cells, and genome-wide off-target analyses established the absence of relevant mutagenic events. When challenging the edited T cells with CCR5-tropic HIV, protection in a dose-dependent manner is observed. Functional assessments reveal no significant differences between edited and control cells in terms of proliferation and their ability to secrete cytokines upon exogenous stimuli. In conclusion, a highly active and specific TALEN to disrupt CCR5 is successfully engineered, paving the way for its clinical application in hematopoietic stem cell grafts.

Silencing of OBP genes: Generation of loss-of-function mutants of PBP by genome editing.

Pheromone binding proteins (PBPs) are small soluble proteins (about 15kDa) that play striking roles in the detection of sex pheromones in insects. Many studies including structural analysis, binding simulation, and in vitro assays have been performed to clarify the modes of action of PBPs. Although these studies have provided valuable contributions toward the understanding of which key amino acid components contribute to the correct folding of PBPs and their binding affinities to sex pheromones, the functional characteristics of PBPs in the natural environment is still obscure. Recent developments in genome editing have begun to enable the functional examination of PBPs in in vivo. Among insect PBPs, BmPBP1 is one of the most well-characterized, there being rich understanding of its structure, biochemical analysis, binding affinity, localization, and the relationship between the type of olfactory receptors and its expression. A recent study has shown that BmPBP1 contributes sensitivity, but not selectivity of sex pheromone detection in the silkmoth Bombyx mori. In this chapter, based on a current report of the functional characterization of BmPBP1 using genome editing, we provide one example of a useful analytical method to clarify the functional role of PBP in vivo.

Characterization of organic anion transporting polypeptide 1b2 knockout rats generated by CRISPR/Cas9: a novel model for drug transport and hyperbilirubinemia disease.

Organic anion transporting polypeptide 1B1 and 1B3 (OATP1B1/3) as important uptake transporters play a fundamental role in the transportation of exogenous drugs and endogenous substances into cells. Rat OATP1B2, encoded by the Slco1b2 gene, is homologous to human OATP1B1/3. Although OATP1B1/3 is very important, few animal models can be used to study its properties. In this report, we successfully constructed the Slco1b2 knockout (KO) rat model via using the CRISPR/Cas9 technology for the first time. The novel rat model showed the absence of OATP1B2 protein expression, with no off-target effects as well as compensatory regulation of other transporters. Further pharmacokinetic study of pitavastatin, a typical substrate of OATP1B2, confirmed the OATP1B2 function was absent. Since bilirubin and bile acids are the substrates of OATP1B2, the contents of total bilirubin, direct bilirubin, indirect bilirubin, and total bile acids in serum are significantly higher in Slco1b2 KO rats than the data of wild-type rats. These results are consistent with the symptoms caused by the absence of OATP1B1/3 in Rotor syndrome. Therefore, this rat model is not only a powerful tool for the study of OATP1B2-mediated drug transportation, but also a good disease model to study hyperbilirubinemia-related diseases.

New Bicistronic TALENs Greatly Improve Genome Editing.

Genome editing has become one of the most powerful tools in present-day stem cell and regenerative medicine research, but despite its rapid acceptance and widespread use, some elements of the technology still need improvement. In this unit, we present data regarding the use of a new, more efficient type of transcription activator-like effector nuclease (TALEN) for gene editing. Our group has generated bicistronic genes in which classical TALEN coding sequences are linked by 2A elements to different reporter molecules, such as fluorochromes (TALEN-F) or membrane receptors (TALEN-M). This structure results in two proteins transcribed from the same transcript, of which the second (the reporter) can be used as the target for selection by fluorescence-assisted cell sorting (FACS) or magnetic-activated cell sorting (MACS). The application of these new TALEN genes allows a rapid enrichment of cells in which both members of the TALEN pair are active, thus eliminating the need for lengthy selection in culture and laborious characterization of a large number of clones. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of new TALENs Basic Protocol 2: Genome editing using TALEN-F Alternate Protocol 1: Generation of TALEN-M Support Protocol 1: mRNA in vitro transcription (IVT) of TALEN-T2A-reporter expression vector Alternate Protocol 2: Editing of primary T cells using TALEN-M Basic Protocol 3: Verifying gene editing Support Protocol 2: Rapid expansion protocol for edited T-cells.

Generation and validation of a PITX2-EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells.

PITX2 (Paired-like homeodomain transcription factor 2) plays important roles in asymmetric development of the internal organs and symmetric development of eye tissues. During eye development, cranial neural crest cells migrate from the neural tube and form the periocular mesenchyme (POM). POM cells differentiate into several ocular cell types, such as corneal endothelial cells, keratocytes, and some ocular mesenchymal cells. In this study, we used transcription activator-like effector nuclease technology to establish a human induced pluripotent stem cell (hiPSC) line expressing a fluorescent reporter gene from the PITX2 promoter. Using homologous recombination, we heterozygously inserted a PITX2-IRES2-EGFP sequence downstream of the stop codon in exon 8 of PITX2 Cellular pluripotency was monitored with alkaline phosphatase and immunofluorescence staining of pluripotency markers, and the hiPSC line formed normal self-formed ectodermal autonomous multizones. Using a combination of previously reported methods, we induced PITX2 in the hiPSC line and observed simultaneous EGFP and PITX2 expression, as indicated by immunoblotting and immunofluorescence staining. PITX2 mRNA levels were increased in EGFP-positive cells, which were collected by cell sorting, and marker gene expression analysis of EGFP-positive cells induced in self-formed ectodermal autonomous multizones revealed that they were genuine POM cells. Moreover, after 2 days of culture, EGFP-positive cells expressed the PITX2 protein, which co-localized with forkhead box C1 (FOXC1) protein in the nucleus. We anticipate that the PITX2-EGFP hiPSC reporter cell line established and validated here can be utilized to isolate POM cells and to analyze PITX2 expression during POM cell induction.

Adeno-Associated Viral Vectors for Homology-Directed Generation of CAR-T Cells.

Immunotherapy with T cells expressing chimeric antigen receptors (CAR) is an emerging and promising treatment against refractory cancers. However, the currently adopted methods of modification of T cells pose a risk of insertional oncogenesis because lentiviral and retroviral vectors integrate the CAR transgene in a semi-random fashion. In addition, this therapy is only available using autologous cells, which create problems in production and limit the access for patients who have their T cells depleted. One modification method that shows the ability to overcome both drawbacks is the knock-in of the CAR simultaneously knocking-out genes that prevent allogeneic therapy, such as the endogenous T cell receptor. In this mini-review, the authors present recent efforts to develop safer universal CAR-T cells. More specifically, the combined application of target-directed nucleases, which create a double-strand break at a specific genome locus, and the delivery of CAR DNA via adeno-associated viral vectors for subsequent integration via homologous recombination and silencing of the targeted gene is focused on.

No excessive mutations in transcription activator-like effector nuclease-mediated α-1,3-galactosyltransferase knockout Yucatan miniature pigs.

OBJECTIVE: Specific genomic sites can be recognized and permanently modified by genome editing. The discovery of endonucleases has advanced genome editing in pigs, attenuating xenograft rejection and cross-species disease transmission. However, off-target mutagenesis caused by these nucleases is a major barrier to putative clinical applications. Furthermore, off-target mutagenesis by genome editing has not yet been addressed in pigs. METHODS: Here, we generated genetically inheritable α-1,3-galactosyltransferase (GGTA1) knockout Yucatan miniature pigs by combining transcription activator-like effector nuclease (TALEN) and nuclear transfer. For precise estimation of genomic mutations induced by TALEN in GGTA1 knockout pigs, we obtained the whole-genome sequence of the donor cells for use as an internal control genome. RESULTS: In-depth whole-genome sequencing analysis demonstrated that TALEN-mediated GGTA1 knockout pigs had a comparable mutation rate to homologous recombination-treated pigs and wild-type strain controls. RNA sequencing analysis associated with genomic mutations revealed that TALEN-induced off-target mutations had no discernable effect on RNA transcript abundance. CONCLUSION: Therefore, TALEN appears to be a precise and safe tool for generating genome-edited pigs, and the TALEN-mediated GGTA1 knockout Yucatan miniature pigs produced in this study can serve as a safe and effective organ and tissue resource for clinical applications.

Biomaterial-based delivery systems of nucleic acid for regenerative research and regenerative therapy.

Regenerative medicine is a new and promising medical method aiming at treating patients with defective or dysfunctional tissues by maintaining or enhancing the biological activity of cells. The development of biomaterial-based technologies, such as cell scaffolds and carriers for drug delivery system, are highly required to promote the regenerative research and regenerative therapy. Nucleic acids are one of the most feasible factors to efficiently modify the biological activity of cells. The effective and stable delivery of nucleic acids into cells is highly required to succeed in the modification. Biomaterials-based non-viral carriers or biological carriers, like exosomes, play an important role in the efficient delivery of nucleic acids. This review introduces the examples of regenerative research and regenerative therapy based on the delivery of nucleic acids with biomaterials technologies and emphasizes their importance to accomplish regenerative medicine.

How farm animals are improving human health and welfare.

The contribution of farm animals to human health and welfare cannot be properly addressed without reflecting on the impact that animal domestication has had upon human civilisation. About 14,000 years ago, the Neolithic revolution started with the domestication of animals and plants, resulting in the emergence of the main agricultural breeds of livestock and crops. In contrast, the breeding of new animal species for biomedical research, such as small rodents and other model species, is a relatively recent activity. The cellular and molecular mechanisms of inheritance have only been understood over the past few decades and translated into approaches to improve breeding success. In recent years, seminal discoveries in the fields of cellular reprogramming, genetic engineering, and whole-genome sequencing have accelerated this development. The first therapeutic proteins produced by biopharming in livestock have been approved to treat human patients. The suitability of pluripotent stem cells as a source for cell replacement therapies is currently being investigated, using farm animals as informative preclinical models. Disease modelling in farm animals allows systematic testing of effective treatments. Within the context of these developments, this concise review will focus on the contribution of farm animals to human health and welfare.

On ne peut traiter de la contribution des animaux d'élevage à la santé et au bienêtre de l'homme sans prendre en compte l'impact de la domestication des animaux sur la civilisation humaine. La révolution néolithique a commencé il y a environ 14 000 ans avec la domestication des animaux et des plantes, ce qui a donné naissance aux principales variétés cultivées et races d'élevage. En revanche, la sélection d'espèces animales nouvelles pour la recherche biomédicale, par exemple certaines espèces de petits rongeurs et d'autres modèles animaux, constitue une activité relativement récente. Ce n'est que depuis quelques dizaines d'années que les mécanismes cellulaires et moléculaires de l'hérédité sont bien compris et appliqués dans des approches permettant d'améliorer le potentiel génétique des élevages. Depuis quelques années, des découvertes fondamentales dans les domaines de la reprogrammation cellulaire, du génie génétique et du séquençage du génome entier ont accéléré cette évolution. Les premières protéines thérapeutiques produites par l'industrie biopharmaceutique chez des animaux d'élevage ont été approuvées pour traiter des patients humains. La recherche examine actuellement les possibilités de recourir à des cellules souches pluripotentes pour mettre en place des thérapies de remplacement, en utilisant des animaux d'élevage comme modèles précliniques. La modélisation des maladies en utilisant des animaux d'élevage permet d'effectuer des essais systématiques de l'efficacité des traitements. Les auteurs consacrent l'essentiel de leur synthèse à la contribution des animaux d'élevage à la santé et au bienêtre de l'homme, dans le cadre de ces évolutions.

No cabe examinar debidamente la contribución de los animales de granja a la salud y el bienestar del ser humano sin detenerse a reflexionar sobre la influencia que ha tenido en la civilización humana la domesticación de los animales. Hace unos 14 000 años, con la domesticación de animales y plantas, dio comienzo la revolución neolítica, que iba a deparar la aparición de las principales razas agrícolas de ganado y cultivos. En marcado contraste, la cría selectiva de nuevas especies animales con fines de investigación biomédica, como pequeños roedores y otras especies utilizadas como modelo, es una actividad relativamente reciente. Solo en los últimos decenios se han desentrañado los mecanismos celulares y moleculares de la herencia y se ha podido traducir este conocimiento en métodos para mejorar los niveles de éxito de la cría selectiva. En los últimos años, esta evolución se ha acelerado gracias a trascendentales descubrimientos en los ámbitos de la reprogramación celular, la ingeniería genética y la secuenciación de genomas completos. Ya están aprobadas las primeras proteínas terapéuticas para tratar a pacientes humanos obtenidas a partir de ganado mediante procedimientos biofarmacéuticos. Actualmente se investiga la idoneidad de las células troncales pluripotentes como fuente de terapias de sustitución celular, utilizando a animales de granja como modelos preclínicos informativos. La modelización de enfermedades en animales de granja permite ensayar tratamientos eficaces de forma sistemática. En el contexto de todos estos adelantos, los autores se centran en repasar concisamente la contribución de los animales de granja a la salud y el bienestar humanos.

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Gene Editing Technique in Xenotransplantation.

Genetically modified pigs have been considered favorable resources in xenotransplantation. Microinjection of randomly integrating transgenes into zygotes, somatic cell nuclear transfer, homologous recombination, zinc finger nucleases, transcription activator-like effector nucleases, and most recently, clustered regularly interspaced short palindromic repeats-cas9 (CRISPR/Cas9) are the techniques that have been used to generate these animals. Here, we provide an overview of the CRISPR approaches that have been used to modify genes which are vital in improving xenograft survival rate, including cytidine monophosphate-N-acetylneuraminic acid hydroxylase, B1,4N-acetylgalactosaminyltransferase, isoglobotrihexosylceramide synthase, class I MHC, von Willebrand factor, C3, and porcine endogenous retroviruses. In addition, we will mention the importance of potential candidate genes which could be targeted using CRISPR/Cas9.

Prion gene paralogs are dispensable for early zebrafish development and have nonadditive roles in seizure susceptibility.

Normally folded prion protein (PrPC) and its functions in healthy brains remain underappreciated compared with the intense study of its misfolded forms ("prions," PrPSc) during the pathobiology of prion diseases. This impedes the development of therapeutic strategies in Alzheimer's and prion diseases. Disrupting the zebrafish homologs of PrPC has provided novel insights; however, mutagenesis of the zebrafish paralog prp2 did not recapitulate previous dramatic developmental phenotypes, suggesting redundancy with the prp1 paralog. Here, we generated zebrafish prp1 loss-of-function mutant alleles and dual prp1-/-;prp2-/- mutants. Zebrafish prp1-/- and dual prp1-/-;prp2-/- mutants resemble mammalian Prnp knockouts insofar as they lack overt phenotypes, which surprisingly contrasts with reports of severe developmental phenotypes when either prp1 or prp2 is knocked down acutely. Previous studies suggest that PrPC participates in neural cell development/adhesion, including in zebrafish where loss of prp2 affects adhesion and deposition patterns of lateral line neuromasts. In contrast with the expectation that prp1's functions would be redundant to prp2, they appear to have opposing functions in lateral line neurodevelopment. Similarly, loss of prp1 blunted the seizure susceptibility phenotypes observed in prp2 mutants, contrasting the expected exacerbation of phenotypes if these prion gene paralogs were serving redundant roles. In summary, prion mutant fish lack the overt phenotypes previously predicted, and instead they have subtle phenotypes similar to mammals. No evidence was found for functional redundancy in the zebrafish prion gene paralogs, and the phenotypes observed when each gene is disrupted individually are consistent with ancient functions of prion proteins in neurodevelopment and modulation of neural activity.

In vitro study of joint intervention of E-cad and Bmi-Ⅰ mediated by transcription activator-like effector nuclease in nasopharyngeal carcinoma / 中南大学学报(医学版)

Objective:To explore the effect of intervention of E-cadherin (E-cad) and B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) mediated by transcription activator-like effector nuclease (TALEN) on the biological behaviors ofnasopharyngeal carcinoma cells.Methods:Multi-locus gene targeting vectors pUC-DS1-CMV-E-cad-2A-Neo-DS2 and pUC-DS1-Bmi-1 shRNA-Zeo-DS2 were constructed,and the E-cad and Bmi-1 targeting vectors were transferred with TALEN plasmids to CNE-2 cells individually or simultaneously.The integration of target genes were detected by PCR,the expressions of E-cad and Bmi-1 were detected by Western blot.The changes of cell proliferation were detected by cell counting kit-8 (CCK-8) assay.T-he cell cycle and apoptosis were detected by flow cytometry.The cell migration and invasion were detected by Transwell assay.Results:The E-cad and Bmi-1 shRNA expression elements were successfully integrated into the genome of CNE-2 cells,the protein expression level of E-cad was up-regulated,and the protein expression level of Bmi-1 was down-regulated.The intervention of E-cad and Bmi-1 didn't affect the proliferation,cell cycle and apoptosis of CNE-2 cells,but it significantly inhibited the migration and invasion ability of CNE-2 cells.Furthermore,the intervention of E-cad and Bmi-1 together significantly inhibited the migration ability of nasopharyngeal carcinoma cells compared with the intervention of E-cad or Bmi-1 alone (all P＜0.01).Conclusion:The joint intervention of E-cad and Bmi-1 mediated by TALEN can effectively inhibit the migration and invasion of nasopharyngeal carcinoma cells in vitro,which may lay the preliminary experimental basis for gene therapy of human cancer.

Advances in genome editing for improved animal breeding: A review.

Since centuries, the traits for production and disease resistance are being targeted while improving the genetic merit of domestic animals, using conventional breeding programs such as inbreeding, outbreeding, or introduction of marker-assisted selection. The arrival of new scientific concepts, such as cloning and genome engineering, has added a new and promising research dimension to the existing animal breeding programs. Development of genome editing technologies such as transcription activator-like effector nuclease, zinc finger nuclease, and clustered regularly interspaced short palindromic repeats systems begun a fresh era of genome editing, through which any change in the genome, including specific DNA sequence or indels, can be made with unprecedented precision and specificity. Furthermore, it offers an opportunity of intensification in the frequency of desirable alleles in an animal population through gene-edited individuals more rapidly than conventional breeding. The specific research is evolving swiftly with a focus on improvement of economically important animal species or their traits all of which form an important subject of this review. It also discusses the hurdles to commercialization of these techniques despite several patent applications owing to the ambiguous legal status of genome-editing methods on account of their disputed classification. Nonetheless, barring ethical concerns gene-editing entailing economically important genes offers a tremendous potential for breeding animals with desirable traits.

Genome editing technologies to fight infectious diseases.

INTRODUCTION: Genome editing by programmable nucleases represents a promising tool that could be exploited to develop new therapeutic strategies to fight infectious diseases. These nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) and homing endonucleases, are molecular scissors that can be targeted at predetermined loci in order to modify the genome sequence of an organism. Areas covered: By perturbing genomic DNA at predetermined loci, programmable nucleases can be used as antiviral and antimicrobial treatment. This approach includes targeting of essential viral genes or viral sequences able, once mutated, to inhibit viral replication; repurposing of CRISPR-Cas9 system for lethal self-targeting of bacteria; targeting antibiotic-resistance and virulence genes in bacteria, fungi, and parasites; engineering arthropod vectors to prevent vector-borne infections. Expert commentary: While progress has been done in demonstrating the feasibility of using genome editing as antimicrobial strategy, there are still many hurdles to overcome, such as the risk of off-target mutations, the raising of escape mutants, and the inefficiency of delivery methods, before translating results from preclinical studies into clinical applications.