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Genome editing has been well established as a genome engineering tool that enables researchers to establish causal linkages between genetic mutation and biological phenotypes, providing further understanding of the genetic manifestation of many debilitating diseases. More recently, the paradigm of genome editing technologies has evolved to include the correction of mutations that cause diseases via the use of nucleases such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and more recently, Cas9 nuclease. With the aim of reversing disease phenotypes, which arise from somatic gene mutations, current research focuses on the clinical translatability of correcting human genetic diseases in vivo, to provide long-term therapeutic benefits and potentially circumvent the limitations of in vivo cell replacement therapy. In this review, in addition to providing an overview of the various genome editing techniques available, we have also summarized several in vivo genome engineering strategies that have successfully demonstrated disease correction via in vivo genome editing. The various benefits and challenges faced in applying in vivo genome editing in humans will also be discussed.

Pigs serve as an important agricultural resource and animal model in biomedical studies. Efficient and precise modification of pig genome by using recently developed gene editing tools has significantly broadened the application of pig models in various research areas. The three types of site-specific nucleases, namely, zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein, are the main gene editing tools that can efficiently introduce predetermined modifications, including knockouts and knockins, into the pig genome. These modifications can confer desired phenotypes to pigs to improve production traits, such as optimal meat production, enhanced feed digestibility, and disease resistance. Besides, given their genetic, anatomic, and physiologic similarities to humans, pigs can also be modified to model human diseases or to serve as an organ source for xenotransplantation to save human lives. To date, many genetically modified pig models with agricultural or biomedical values have been established by using gene editing tools. These pig models are expected to accelerate research progress in related fields and benefit humans.

Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations.

This special issue of Chromosome Research contains 12 invited topical review articles, which cover some of the most active areas of genome engineering using artificial chromosomes and engineered nucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9. This new field continues to be rapidly developed and, indeed, in the past year alone, there have been 513 papers cited in PubMed that are focused on genome/chromosome engineering in different organisms ranging from bacteria to plants and animals, as well as in cultured cells such as ES and iPS cells. These review articles were written by experts of their respective fields to bring together a vast amount of literature in a clear and didactic manner.

The β-thalassemias are a group of hereditary hematological diseases caused by over 300 mutations of the adult β-globin gene. Together with sickle cell anemia, thalassemia syndromes are among the most impactful diseases in developing countries, in which the lack of genetic counseling and prenatal diagnosis have contributed to the maintenance of a very high frequency of these genetic diseases in the population. Gene therapy for β-thalassemia has recently seen steadily accelerating progress and has reached a crossroads in its development. Presently, data from past and ongoing clinical trials guide the design of further clinical and preclinical studies based on gene augmentation, while fundamental insights into globin switching and new technology developments have inspired the investigation of novel gene-therapy approaches. Moreover, human erythropoietic stem cells from β-thalassemia patients have been the cellular targets of choice to date whereas future gene-therapy studies might increasingly draw on induced pluripotent stem cells. Herein, we summarize the most significant developments in β-thalassemia gene therapy over the last decade, with a strong emphasis on the most recent findings, for β-thalassemia model systems; for β-, γ-, and anti-sickling β-globin gene addition and combinatorial approaches including the latest results of clinical trials; and for novel approaches, such as transgene-mediated activation of γ-globin and genome editing using designer nucleases.

PKD1 and PKD2 mutations could lead to autosomal dominant polycystic kidney disease (ADPKD), which afflicts millions of people worldwide. Due to the marked differences in the lifespan, size, anatomy, and physiology from humans, rodent ADPKD models cannot fully mimic the disease. To obtain a large animal model that recapitulates the disease, we constructed a mini-pig model by mono-allelic knockout (KO) of PKD1 using zinc finger nuclease. The mono-allelic KO pigs had lower PKD1 expression than their wild-type littermates at both the transcriptional and translational levels. After approximately six months, renal cysts appeared and grew progressively in the KO pigs. Histological analysis showed that renal cysts were scatteredly distributed in the mutant pig kidneys and were lined by either cuboidal or flattened epithelial cells. Contrast-enhanced computed tomography confirmed that all of the mutant pigs had renal and hepatic cysts, when they were 11-month-old. Immunohistochemical analysis revealed that most of the cysts were derived from the proximal tubules and collecting ducts. Therefore, the PKD1 mono-allelic knockout is sufficient to trigger renal cystogenesis, and this pig model may provide a platform for future study of renal cyst formation.

Breakthroughs in the development of programmable site-specific nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases (MNs), and most recently, the clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (including Cas9) have greatly enabled and accelerated genome editing. By targeting double-strand breaks to user-defined locations, the rates of DNA repair events are greatly enhanced relative to un-catalyzed events at the same sites. However, the underlying biology of each genome-editing nuclease influences the targeting potential, the spectrum of off-target cleavages, the ease-of-use, and the types of recombination events at targeted double-strand breaks. No single genome-editing nuclease is optimized for all possible applications. Here, we focus on the diversity of nuclease domains available for genome editing, highlighting biochemical properties and the potential applications that are best suited to each domain.

Programmable endonucleases introduce DNA breaks at specific sites, which are repaired by non-homologous end joining (NHEJ) or homology recombination (HDR). Genome editing in human lymphoid cells is challenging as these difficult-to-transfect cells may also inefficiently repair DNA by HDR. Here, we estimated efficiencies and dynamics of knockout (KO) and knockin (KI) generation in human T and B cell lines depending on repair template, target loci and types of genomic endonucleases. Using zinc finger nuclease (ZFN), we have engineered Jurkat and CEM cells with the 8.2 kb human immunodeficiency virus type 1 (HIV-1) ∆Env genome integrated at the adeno-associated virus integration site 1 (AAVS1) locus that stably produce virus particles and mediate infection upon transfection with helper vectors. Knockouts generated by ZFN or clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) double nicking techniques were comparably efficient in lymphoid cells. However, unlike polyclonal sorted cells, gene-edited cells selected by cloning exerted tremendous deviations in functionality as estimated by replication of HIV-1 and human T cell leukemia virus type 1 (HTLV-1) in these cells. Notably, the recently reported high-fidelity eCas9 1.1 when combined to the nickase mutation displayed gene-dependent decrease in on-target activity. Thus, the balance between off-target effects and on-target efficiency of nucleases, as well as choice of the optimal method of edited cell selection should be taken into account for proper gene function validation in lymphoid cells.

On May 11th and 12th 2017 was held in Nantes, France, the international meeting “Advances in transgenic animal models and techniques” ( http://www.trm.univ-nantes.fr/ ). This biennial meeting is the fifth one of its kind to be organized by the Transgenic Rats ImmunoPhenomic (TRIP) Nantes facility ( http://www.tgr.nantes.inserm.fr/ ). The meeting was supported by private companies (SONIDEL, Scionics computer innovation, New England Biolabs, MERCK, genOway, Journal Disease Models and Mechanisms) and by public institutions (International Society for Transgenic Technology, University of Nantes, INSERM UMR 1064, SFR Francois Bonamy, CNRS, Region Pays de la Loire, Biogenouest, TEFOR infrastructure, ITUN, IHU-CESTI and DHU-Oncogeffe and Labex IGO). Around 100 participants, from France but also from different European countries, Japan and USA, attended the meeting.

Summary Tauopathies such as frontotemporal dementia (FTD) remain incurable to date, partially due to the lack of translational in vitro disease models. The MAPT gene, encoding the microtubule-associated protein tau, has been shown to play an important role in FTD pathogenesis. Therefore, we used zinc finger nucleases to introduce two MAPT mutations into healthy donor induced pluripotent stem cells (iPSCs). The IVS10+16 mutation increases the expression of 4R tau, while the P301S mutation is pro-aggregant. Whole-transcriptome analysis of MAPT IVS10+16 neurons reveals neuronal subtype differences, reduced neural progenitor proliferation potential, and aberrant WNT/SHH signaling. Notably, these neurodevelopmental phenotypes could be recapitulated in neurons from patients carrying the MAPT IVS10+16 mutation. Moreover, the additional pro-aggregant P301S mutation revealed additional phenotypes, such as an increased calcium burst frequency, reduced lysosomal acidity, tau oligomerization, and neurodegeneration. This series of iPSCs could serve as a platform to unravel a potential link between pathogenic 4R tau and FTD., Highlights • Analysis of ZFN-engineered MAPT IVS10+16 with or without additional P301S mutation • Neurodevelopmental phenotypes in ZFN and patient-derived MAPT IVS10+16 neurons • Neurodegenerative phenotypes in MAPT IVS10+16/P301S double-mutant neurons, In this article, Verheyen and colleagues introduced 2 MAPT mutations (IVS10+16/P301S) into healthy donor iPSCs. Characterization of MAPT IVS10+16 neurons revealed neurodevelopmental neuron subtype differences, reduced neural progenitor proliferation, and aberrant WNT/SHH signaling, while the additional pro-aggregant P301S mutation revealed phenotypes more related to neurodegeneration. These iPSCs could help to unravel a potential link between pathogenic 4R tau and FTD.

Hemophilia A and B are congenital, X-linked bleeding disorders caused by mutations in the genes encoding for the blood clotting factor VIII (FVIII) or factor IX (FIX), respectively. Since the beginning of gene therapy, hemophilia has been considered an attractive disease target that served as a trailblazer for the field at large. Different technologies have been explored to efficiently and safely deliver the therapeutic FVIII and FIX genes into the patients' cells. Currently, the most promising vectors for hemophilia gene therapy are adeno-associated viral vectors (AAVs) and lentiviral vectors. More recently, gene editing approaches based on designer nucleases or CRISPR/Cas, have also been considered to minimize risks associated with random vector integration and insertional mutagenesis though off-target issues would have to be carefully and comprehensively assessed. In the past two decades, several phase 1 hemophilia gene therapy clinical trials have been initiated with varying success. In particular, the early gene therapy clinical trials in hemophilia B patients based on AAV showed either transient or subtherapeutic clotting factor expression levels. This could be ascribed, at least in part, to suboptimal vector design and/or inadvertent immune consequences triggering hepatic inflammation. Hence, there was an unmet need to further increase vector safety and efficacy in future trials, preferably by using lower vector doses. It is particularly encouraging that sustained therapeutic FVIII and FIX expression levels have recently been attained after gene therapy in patients with severe hemophilia paving the way towards pivotal trials and commercialization. Nevertheless, transient liver toxicity still occurs and the use of transient immunosuppression was still required to curtail inadvertent immune responses, especially at high vector doses. To further boost clotting factor expression levels, codon-usage optimized synthetic FVIII or FIX transgenes have been employed. Alternatively, we and others have shown that the incorporation of hyperactive gain-of-function R338L mutation in the FIX gene substantially increased the overall efficacy. It is inevitable that the continued improvements in vector engineering and new insights in the vector-patient interactions will further benefit the development of a safe and effective cure for hemophilia A and B.

Mouse transgenesis has been instrumental in determining the function of genes in the pathophysiology of human diseases and modification of genes by homologous recombination in mouse embryonic stem cells remains a widely used technology. However, this approach harbors a number of disadvantages, as it is time-consuming and quite laborious. Over the last decade a number of new genome editing technologies have been developed, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas). These systems are characterized by a designed DNA binding protein or RNA sequence fused or co-expressed with a non-specific endonuclease, respectively. The engineered DNA binding protein or RNA sequence guides the nuclease to a specific target sequence in the genome to induce a double strand break. The subsequent activation of the DNA repair machinery then enables the introduction of gene modifications at the target site, such as gene disruption, correction or insertion. Nuclease-mediated genome editing has numerous advantages over conventional gene targeting, including increased efficiency in gene editing, reduced generation time of mutant mice, and the ability to mutagenize multiple genes simultaneously. Although nuclease-driven modifications in the genome are a powerful tool to generate mutant mice, there are concerns about off-target cleavage, especially when using the CRISPR/Cas system. Here, we describe the basic principles of these new strategies in mouse genome manipulation, their inherent advantages, and their potential disadvantages compared to current technologies used to study gene function in mouse models. This article is part of a Special Issue entitled: From Genome to Function. (C) 2014 Elsevier B.V. All rights reserved.

Mouse transgenesis has been instrumental in determining the function of genes in the pathophysiology of human diseases and modification of genes by homologous recombination in mouse embryonic stem cells remains a widely used technology. However, this approach harbors a number of disadvantages, as it is time-consuming and quite laborious. Over the last decade a number of new genome editing technologies have been developed, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas). These systems are characterized by a designed DNA binding protein or RNA sequence fused or co-expressed with a non-specific endonuclease, respectively. The engineered DNA binding protein or RNA sequence guides the nuclease to a specific target sequence in the genome to induce a double strand break. The subsequent activation of the DNA repair machinery then enables the introduction of gene modifications at the target site, such as gene disruption, correction or insertion. Nuclease-mediated genome editing has numerous advantages over conventional gene targeting, including increased efficiency in gene editing, reduced generation time of mutant mice, and the ability to mutagenize multiple genes simultaneously. Although nuclease-driven modifications in the genome are a powerful tool to generate mutant mice, there are concerns about off-target cleavage, especially when using the CRISPR/Cas system. Here, we describe the basic principles of these new strategies in mouse genome manipulation, their inherent advantages, and their potential disadvantages compared to current technologies used to study gene function in mouse models. This article is part of a Special Issue entitled: From Genome to Function. (C) 2014 Elsevier B.V. All rights reserved.

In the decade since Yamanaka and colleagues described methods to reprogram somatic cells into a pluripotent state, human induced pluripotent stem cells (hiPSCs) have demonstrated tremendous promise in numerous disease modeling, drug discovery, and regenerative medicine applications. More recently, the development and refinement of advanced gene transduction and editing technologies have further accelerated the potential of hiPSCs. In this review, we discuss the various gene editing technologies that are being implemented with hiPSCs. Specifically, we describe the emergence of technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 that can be used to edit the genome at precise locations, and discuss the strengths and weaknesses of each of these technologies. In addition, we present the current applications of these technologies in elucidating the mechanisms of human development and disease, developing novel and effective therapeutic molecules, and engineering cell-based therapies. Finally, we discuss the emerging technological advances in targeted gene editing methods.

Plant-virus interactions based-studies have contributed to increase our understanding on plant resistance mechanisms, providing new tools for crop improvement. In the last two decades, RNA interference (RNAi), a post-transcriptional gene silencing approach, has been used to induce antiviral defences in plants with the help of genetic engineering technologies. More recently, the new genome editing systems (GES) are revolutionizing the scope of tools available to confer virus resistance in plants. The most explored GES are Zinc finger nucleases (ZFN), Transcription activator-like effector nucleases (TALEN) and Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 endonuclease. GES are engineered to target and introduce mutations, which can be deleterious, via double-strand breaks at specific DNA sequences by the error-prone non-homologous recombination end-joining pathway. Although GES have been engineered to target DNA, recent discoveries of GES targeting ssRNA molecules, including virus genomes, pave the way for further studies programming plant defence against RNA viruses. Most of plant virus species have an RNA genome and at least 784 species have positive ssRNA. Here, we provide a summary of the latest progress in plant antiviral defences mediated by GES. In addition, we also discuss briefly the GES perspectives in light of the rebooted debate on genetic modified organisms (GMOs) and the current regulatory frame for agricultural products involving the use of such engineering technologies.

The goal of editing the genomes of stem cells to generate model organisms and cell lines for genetic and biological studies has been pursued for decades. There is also exciting potential for future clinical impact in humans. While recent, rapid advances in targeted nuclease technologies have led to unprecedented accessibility and ease of gene editing, biology has benefited from past directed gene modification via homologous recombination, gene traps and other transgenic methodologies. Here we review the history of genome editing in stem cells (including via zinc finger nucleases, transcription activator-like effector nucleases and CRISPR-Cas9), discuss recent developments leading to the implementation of stem cell gene therapies in clinical trials and consider the prospects for future advances in this rapidly evolving field.

International audience; Genetically engineered sheep and goats represent useful models applied to proof of concepts, large-scale production of novel products or processes, and improvement of animal traits, which is of interest in biomedicine, biopharma, and livestock. This disruptive biotechnology arose in the 80s by injecting DNA fragments into the pronucleus of zygote-staged embryos. Pronuclear microinjection set the transgenic concept into people's mind but was characterized by inefficient and often frustrating results mostly because of uncontrolled and/or random integration and unpredictable transgene expression. Somatic cell nuclear transfer launched the second wave in the late 90s, solving several weaknesses of the previous technique by making feasible the transfer of a genetically modified and fully characterized cell into an enucleated oocyte, capable of cell reprogramming to generate genetically engineered animals. Important advances were also achieved during the 2000s with the arrival of new techniques like the lentivirus system, transposons, RNA interference, site-specific recombinases, and sperm-mediated transgenesis. We are now living the irruption of the third technological wave in which genome edition is possible by using endonucleases, particularly the CRISPR/Cas system. Sheep and goats were recently produced by CRISPR/Cas9, and for sure, cattle will be reported soon. We will see new genetically engineered farm animals produced by homologous recombination, multiple gene editing in one-step generation and conditional modifications, among other advancements. In the following decade, genome edition will continue expanding our technical possibilities, which will contribute to the advancement of science, the development of clinical or commercial applications, and the improvement of people's life quality around the world.

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell's endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent stem cells.

Accurate and efficient genome editing is primarily dependent on the generation of a sequence-specific, genomic double-stranded DNA break (DSB) combined with the introduction of an exogenous DNA template into target cells. The exogenous template, called donor DNA, normally contains the foreign sequences flanked by DNA regions sharing sequence identity ("homologous") to those bracketing the target site. The strategies for mediating the formation of DSBs at the predefined genomic loci, have been undergoing intense investigation since the introduction of sequence-customizable zinc-finger nuclease (ZFN) technology. More recently, prokaryotic protein-based transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases (RGNs) derived from CRISPR-associated protein (Cas9) complexes have substantially broadened the availability and applicability of designer nuclease-mediated genome editing. A potential alternative research line to the use of designer nucleases, is to investigate whether specific DNA structures can, by themselves, serve as triggers of the DNA damage response and, in doing so, elicit targeted gene repair. Such an approach would simplify genome editing protocols, such as, by reducing the number of reagents needed to be introduced into target cells. In this thesis, the roles of these secondary structures as well as designer nucleases and donor-DNA templates, delivered via adenoviral vectors, is described.

Recent advancement in genome engineering technology is changing the landscape of biological research and providing neuroscientists with an opportunity to develop new methodologies to ask critical research questions. This advancement is highlighted by the increased use of programmable DNA-binding agents (PDBAs) such as transcription activator-like effector (TALE) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems. These PDBAs fused or co-expressed with various effector domains allow precise modification of genomic sequences and gene expression levels. These technologies mirror and extend beyond classic gene targeting methods contributing to the development of novel tools for basic and clinical neuroscience. In this Review, we discuss the recent development in genome engineering and potential applications of this technology in the field of neuroscience.

Glycogen storage disease type Ia is an autosomal recessive disorder caused by a mutation in the glucose-6-phosphatase (G6Pase) catalytic subunit, encoded in humans by G6PC. G6Pase dephosphorylates glucose-6-phosphate (G6P) in the liver to generate glucose that can be shuttled to the bloodstream to maintain normoglycemia. Patients with GSD Ia typically present at 6 months of age with sever hypoglycemia, which is lethal if untreated. The current treatment is a strict dietary regimen in which children must be fed every 2 hours overnight or given nasogastric tube feeding, and adults must consume uncooked cornstarch around the clock to maintain normal blood sugar levels. This treatment maintains survival but fails to prevent other symptoms related to metabolism of the excess G6P, and patients develop hepatic adenomas that may become hepatocellular carcinoma later in life, in addition to progressive renal complications.To overcome the problems persisting during dietary therapy, the Koeberl lab has sought to develop gene therapy approaches that use adeno-associated virus (AAV) vectors to replace the G6pase activity, restoring normoglycemia and normal metabolic processes. However, the vast majority of AAV-delivered genetic material exists as episomes that do not replicate as cells divide, so the effects of AAV gene therapy on GSD Ia mouse and dog models have proven temporary. We hypothesized that driving integration of therapeutic vector genomes into an affected individual's genome would improve beneficial effects' longevity.We tested several approaches to accomplish this, and have found positive effects using a zinc finger nuclease (ZFN) that targets the mouse safe harbor ROSA26 locus to induce homologous recombination of the G6PC donor vector into the mouse genome. We were able to see an improvement in mouse survival to 8 months of age, an increase in G6Pase activity at 3 months of age, and a decrease in glycogen accumulation at 3 months of age, when the ZFN vector is administered alongside the G6PC vector, compared with mice that received the G6PC vector alone.We have also taken an alternative approach to overcoming the long-term complications of the current dietary treatment, which would augment rather than replace the current treatment. We have examined several drugs known to induce autophagy in other disease models or cell culture systems, to determine if we could manipulate autophagic activity in G6PC knockdown hepatocytes or GSD Ia mice. We have found positive results using rapamycin, a well-studied MTOR inhibitor, in mice and cells, and have screened several other drugs as well, finding positive effects for bezafibrate, mifepristone, carbamazepin, and lithium chloride, in terms of lipid reduction (which accumulates as a symptom of GSD Ia) and/or LC3-II enhancement, which is reduced in GSD Ia due to downregulation of autophagy during G6P accumulation.

Small nonprotein-coding microRNAs (miRNAs) are present in most eukaryotes and are central effectors of RNA silencing-mediated mechanisms for gene expression regulation. In plants, DICER-LIKE1 (DCL1) is the founding member of a highly conserved family of RNase III-like endonucleases that function as core machinery proteins to process hairpin-like precursor transcripts into mature miRNAs, small regulatory RNAs, 21–22 nucleotides in length. Zinc finger nucleases (ZFNs) were used to generate single and double-mutants of putative soybean DCL1 homologs, DCL1a and DCL1b, to confirm their functional role(s) in the soybean miRNA pathway. Neither DCL1 single mutant, dcl1a or dcl1b plants, exhibited a pronounced morphological or molecular phenotype. However, the dcl1a/dcl1b double mutant expressed a strong morphological phenotype, characterized by reduced seed size and aborted seedling development, in addition to defective miRNA precursor transcript processing efficiency and deregulated miRNA target gene expression. Together, these findings indicate that the two soybean DCL1 paralogs, DCL1a and DCL1b, largely play functionally redundant roles in the miRNA pathway and are essential for normal plant development.

Within the last years numerous publications successfully applied sequence specific designer nucleases for genome editing in human PSCs. However, despite this abundance of reports together with the rapid development and improvement accomplished with the technology, it is still difficult to choose the optimal methodology for a specific application of interest. With focus on the most suitable approach for specific applications, we present a practical guidance for successful gene editing in human PSCs using designer nucleases. We discuss experimental considerations, limitations and critical aspects which will guide the investigator for successful implementation of this technology.

The cornea represents an ideal tissue to elucidate the potential of molecular medicine to treat genetic disorders. It is a small,readily accessible tissue that is easy to monitor treat topically, while a number of corneal disorders are well characterizedwith regard to a known genetic cause. In addition, due to their genetic basis most of these conditions are bilateral, affecting both corneas, allowing for a clear comparison between treated and untreated tissue using the fellow eye as a control.Currently, molecular techniques utilising siRNAs, CRISPR/Cas9, TALENS and ZFNs are being evaluated by many for theirtherapeutic potential for corneal disorders. Methodologies are discussed within this review with particular emphasis on themore recent and emerging field of CRISPR/Cas9-based therapeutics. Since the advent of gene therapies, effective deliveryhas been a challenge. Potential ocular delivery techniques include viral vectors, nanoparticles or physical injection.Studies employing in vitro and in vivo models of corneal disorders have demonstrated promising results for treatment by moleculartechniques. However, many of these studies have not translated into the clinic, perhaps due to a lack of good animal models for preclinical testing or failure to develop suitable delivery methods. Until this occurs it is difficult to fully gauge theusefulness of these techniques to treat corneal disorders in human patients over a sustained time period. Here we present a review of current research into molecular-based therapies for genetic corneal disorders and discuss theapplicability of these studies to advancing translational research for other genetic disorders.

The recent development of gene editing tools and methodology for use in livestock enables the production of new animal disease models. These tools facilitate site‐specific mutation of the genome, allowing animals carrying known human disease mutations to be produced. In this review, we describe the various gene editing tools and how they can be used for a range of large animal models of diseases. This genomic technology is in its infancy but the expectation is that through the use of gene editing tools we will see a dramatic increase in animal model resources available for both the study of human disease and the translation of this knowledge into the clinic. Comparative pathology will be central to the productive use of these animal models and the successful translation of new therapeutic strategies. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Background Practical approaches for multigene transformation and gene stacking are extremely important for engineering complex traits and adding new traits in transgenic crops. Trait deployment by gene stacking would greatly simplify downstream plant breeding and trait introgression into cultivars. Gene stacking into pre-determined genomic sites depends on mechanisms of targeted DNA integration and recycling of selectable marker genes. Targeted integrations into chromosomal breaks, created by nucleases, require large transformation efforts. Recombinases such as Cre-lox, on the other hand, efficiently drive site-specific integrations in plants. However, the reversibility of Cre-lox recombination, due to the incorporation of two cis-positioned lox sites, presents a major bottleneck in its application in gene stacking. Here, we describe a strategy of resolving this bottleneck through excision of one of the cis-positioned lox, embedded in the marker gene, by nuclease activity. Methods All transgenic lines were developed by particle bombardment of rice callus with plasmid constructs. Standard molecular approach was used for building the constructs. Transgene loci were analyzed by PCR, Southern hybridization, and DNA sequencing. Results We developed a highly efficient gene stacking method by utilizing powerful recombinases such as Cre-lox and FLP-FRT, for site-specific gene integrations, and nucleases for marker gene excisions. We generated Cre-mediated site-specific integration locus in rice and showed excision of marker gene by I-SceI at ~20 % efficiency, seamlessly connecting genes in the locus. Next, we showed ZFN could be used for marker excision, and the locus can be targeted again by recombinases. Hence, we extended the power of recombinases to gene stacking application in plants. Finally, we show that heat-inducible I-SceI is also suitable for marker excision, and therefore could serve as an important tool in streamlining this gene stacking platform. Conclusions A practical approach for gene stacking in plant cell was developed that allows targeted gene insertions through rounds of transformation, a method needed for introducing new traits into transgenic lines for their rapid deployment in the field. By using Cre-lox, a powerful site-specific recombination system, this method greatly improves gene stacking efficiency, and through the application of nucleases develops marker-free, seamless stack of genes at pre-determined chromosomal sites.

Severe combined immunodeficiency (SCID) and other severe non-SCID primary immunodeficiencies (non-SCID PID) can be treated by allogeneic hematopoietic stem cell (HSC) transplantation, but when histocompatibility leukocyte antigen-matched donors are lacking, this can be a high-risk procedure. Correcting the patient’s own HSCs with gene therapy offers an attractive alternative. Gene therapies currently being used in clinical settings insert a functional copy of the entire gene by means of a viral vector. With this treatment, severe complications may result due to integration within oncogenes. A promising alternative is the use of endonucleases such as ZFNs, TALENs, and CRISPR/Cas9 to introduce a double-stranded break in the DNA and thus induce homology-directed repair. With these genome-editing tools a correct copy can be inserted in a precisely targeted “safe harbor.” They can also be used to correct pathogenic mutations in situ and to develop cellular or animal models needed to study the pathogenic effects of specific genetic defects found in immunodeficient patients. This review discusses the advantages and disadvantages of these endonucleases in gene correction and modeling with an emphasis on CRISPR/Cas9, which offers the most promise due to its efficacy and versatility.

International audience; Trinucleotide repeat expansions are involved in more than two dozen neurological and developmental disorders. Conventional therapeutic approaches aimed at regulating the expression level of affected genes, which rely on drugs, oligonucleotides, and/or transgenes, have met with only limited success so far. An alternative approach is to shorten repeats to non-pathological lengths using highly specific nucleases. Here, I review early experiments using meganucleases, zinc-finger nucleases (ZFN), and transcription-activator like effector nucleases (TALENs) to contract trinucleotide repeats, and discuss the possibility of using CRISPR-Cas nucleases to the same end. Although this is a nascent field, I explore the possibility of designing nucleases and effectively delivering them in the context of gene therapy.