Plants, animals and human genes that are successfully targeted using ZFN, which demonstrates the generality of this approach As of 2012 efficient<unk>  had been developed for a wide range of experimental systems ranging from plants to animals, often beyond clinical interest, and was becoming a standard experimental strategy in research labs. The recent generation of rat, zebrafish, maize and tobacco ZFN-mediated mutants and the improvements in TALEN-based approaches testify to the significance of the methods, and the list is expanding rapidly.<unk>  with engineered nucleases will likely contribute to many fields of life sciences from studying gene functions in plants and animals to gene therapy in humans. For instance, the field of synthetic biology which aims to engineer cells and organisms to perform novel functions, is likely to benefit from the ability of engineered nuclease to add or remove<unk> c elements and therefore create complex systems. In addition, gene functions can be studied using stem cells with engineered nucleases. Listed below are some specific tasks this method can carry out: * Targeted gene mutation * Gene therapy * Creating chromosome rearrangement * Study gene function with stem cells * Transgenic animals * Endogenous gene labeling * Targeted transgene addition Targeted gene modification in animals The combination of recent discoveries in genetic engineering, particularly gene<unk>  and the latest improvement in bovine reproduction technologies (e.g. in vitro embryo culture) allows for<unk>  directly in fertilised oocytes using synthetic highly specific endonucleases. RNA-guided endonucleases:clustered regularly interspaced short palindromic repeats associated Cas9 (CRISPR/Cas9) are a new tool, further increasing the range of methods available. In particular CRISPR/Cas9 engineered endonucleases allows the use of multiple guide RNAs for simultaneous Knockouts (KO) in one step by cytoplasmic direct injection (CDI) on mammalian zygotes. Furthermore, gene<unk>  can be applied to certain types of fish in aquaculture such as Atlantic salmon. Gene<unk>  in fish is currently experimental, but the possibilities include growth, disease resistance, sterility, controlled reproduction, and colour. Selecting for these traits can allow for a more sustainable environment and better welfare for the fish. AquAdvantage salmon is a genetically modified Atlantic salmon developed by AquaBounty Technologies. The growth hormone-regulating gene in the Atlantic salmon is replaced with the growth hormone-regulating gene from the Pacific Chinook salmon and a promoter sequence from the ocean pout Thanks to the parallel development of single-cell transcriptomics,<unk>  and new stem cell models we are now entering a scientifically exciting period where functional genetics is no longer restricted to animal models but can be performed directly in human samples. Single-cell gene expression analysis has resolved a transcriptional road-map of human development from which key candidate genes are being identified for functional studies. Using global transcriptomics data to guide experimentation, the CRISPR based<unk>  tool has made it feasible to disrupt or remove key genes in order to elucidate function in a human setting. Targeted gene modification in plants Overview of GEEN workflow and<unk>  possibilities<unk>  using Meganuclease, ZFNs, and TALEN provides a new strategy for genetic manipulation in plants and are likely to assist in the engineering of desired plant traits by modifying endogenous genes. For instance, site-specific gene addition in major crop species can be used for 'trait stacking' whereby several desired traits are physically linked to ensure their co-segregation during the breeding processes. Progress in such cases have been recently reported in Arabidopsis thaliana and Zea mays. In Arabidopsis thaliana, using ZFN-assisted gene targeting, two herbicide-resistant genes (tobacco acetolactate synthase SuRA and SuRB) were introduced to SuR loci with as high as 2% transformed cells with mutations. In Zea mays, disruption of the target locus was achieved by ZFN-induced DSBs and the resulting NHEJ. ZFN was also used to drive herbicide-tolerance gene expression cassette (PAT) into the targeted endogenous locus IPK1 in this case. Such<unk>  modification observed in the regenerated plants has been shown to be inheritable and was transmitted to the next generation. A potentially successful example of the application of<unk>  techniques in crop improvement can be found in banana, where scientists used CRISPR/Cas9<unk>  to inactivate the endogenous banana streak virus in the B<unk>  of banana (Musa spp.) to overcome a major challenge in banana breeding. In addition, TALEN-based<unk>  engineering has been extensively tested and optimized for use in plants. TALEN fusions have also been used by a U.S. food ingredient company, Calyxt, to improve the quality of soybean oil products and to increase the storage potential of potatoes Several optimizations need to be made in order to improve<unk>  plant<unk> s using ZFN-mediated targeting. There is a need for reliable design and subsequent test of the nucleases, the absence of toxicity of the nucleases, the appropriate choice of the plant tissue for targeting, the routes of induction of enzyme activity, the lack of off-target mutagenesis, and a reliable detection of mutated cases. A common delivery method for CRISPR/Cas9 in plants is Agrobacterium-based transformation. T-DNA is introduced directly into the plant<unk>  by a T4SS mechanism. Cas9 and gRNA-based expression cassettes are turned into Ti plasmids, which are transformed in Agrobacterium for plant application. To improve Cas9 delivery in live plants, viruses are being used more effective transgene delivery.
Genome editing
Gene delivery
Hair cloning
Human cloning
Gene doping
Therapeutic gene modulation
Gene therapy
Osseoincorporation