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The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease using a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications1–5. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show this enhanced TALEN toolkit demonstrates a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino (MO)-based targeted gene knockdowns6. With this updated TALEN system, we successfully used single-stranded DNA (ssDNA) oligonucleotides (oligos) to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair (HDR), including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.

BackgroundRecent studies of transcription activator-like (TAL) effector domains fused to nucleases (TALENs) demonstrate enormous potential for genome editing. Effective design of TALENs requires a combination of selecting appropriate genetic features, finding pairs of binding sites based on a consensus sequence, and, in some cases, identifying endogenous restriction sites for downstream molecular genetic applications.ResultsWe present the web-based program Mojo Hand for designing TAL and TALEN constructs for genome editing applications (http://www.talendesign.org). We describe the algorithm and its implementation. The features of Mojo Hand include (1) automatic download of genomic data from the National Center for Biotechnology Information, (2) analysis of any DNA sequence to reveal pairs of binding sites based on a user-defined template, (3) selection of restriction-enzyme recognition sites in the spacer between the TAL monomer binding sites including options for the selection of restriction enzyme suppliers, and (4) output files designed for subsequent TALEN construction using the Golden Gate assembly method.ConclusionsMojo Hand enables the rapid identification of TAL binding sites for use in TALEN design. The assembly of TALEN constructs, is also simplified by using the TAL-site prediction program in conjunction with a spreadsheet management aid of reagent concentrations and TALEN formulation. Mojo Hand enables scientists to more rapidly deploy TALENs for genome editing applications.

IntroductionAcute myeloid leukemia (AML) refers to a genetically and biologically heterogeneous group of diseases characterized by an abnormal increase of myeloblasts in the bone marrow (BM) and peripheral blood (PB) circulation. Chemotherapy and hematopoietic stem cell transplantation (HSCT) are the mainstays of treatment, but these modalities have reached an impasse with an overall cure rate of only 30%-40%. 1 An attractive strategy is to target specific genetic and biochemical alterations in AML, thereby providing an alternative treatment modality that may improve patient outcome. 2,3 FLT3 (fms-like tyrosine kinase-3) is a receptor tyrosine kinase (RTK) that is highly expressed in hematopoietic stem and progenitor cells. It includes an extracellular domain (ECD), a transmembrane domain (TMD), a juxtamembrane domain (JMD), and 2 tyrosine kinase domains (TKDs) separated by a kinase insert. 4 On binding with FLT3 ligand secreted by BM stromal cells, FLT3 undergoes dimerization, phosphorylation, and TKD activation. The FLT3 gene is mutated in approximately 30% of AMLs, particularly those with normal karyotypes, t(6;9), t(15;17), or trisomy 8. 5,6 The most common mutation is an internal tandem duplication (ITD) up to a few hundred base-pairs within the JMD. Single-base mutations have also been described, most commonly resulting in a substitution of aspartic acid with tyrosine or less commonly a histidine at residue 835 in the TKD. 7,8 At a molecular level, these mutations result in constitutive activation of the FLT3 receptor and hence downstream PI3K/AKT/mTOR, Ras/Raf/MEK/ERK, and JAK/ STAT pathways. 9,10 The biologic consequences are enhanced proliferation and reduced apoptosis of the myeloblasts, which contribute to leukemogenesis. [11][12][13][14][15] Patients with FLT3-ITD respond poorly to conventional chemotherapy and have an inferior prognosis, 16,17 particularly in those with a high FLT3-ITD ϩ cell burden, 18 long ITD sequences, 19 and multiple FLT3-ITD ϩ subclones, 20 underscoring a pathogenetic role of FLT3-ITD in human AML. In mice, knock-in of a heterozygous FLT3-ITD resulted in a preleukemic model of a myeloproliferative disease, providing an in vivo demonstration of the important role of FLT3 in leukemia initiation. 21 We 22 and others 23,24 have shown that the FLT3-ITD allele can be found in leukemia initiating cells (LICs), as distinguished by their capability of regenerating leukemic progeny in transplanted immunodeficient mice. Therefore, targeting FLT3-ITD might provide a novel approach to therapeutic intervention.Several clinical trials on multi-TK inhibitors with different FLT3 specificities and in vitro efficacies have been reported, including the use of midostaurin, 25 lestautinib, 26 tandutinib, 27 sunitinib, 28 and sorafenib. 29 In most studies, clinical efficacy was restricted to FLT3-ITD ϩ AML and correlated with inactivation of FLT3 phosphorylation. 25,[30][31][32] Complete remission was rare and limited to anecdotal reports in relapsed AML after allogeneic HSCT. 33 Furthermore, after ...

One key problem in precision genome editing is the unpredictable plurality of sequence outcomes at the site of targeted DNA double stranded breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of the identical alleles using NHEJ is a labor intensive process. In this study, we propose Microhomology-mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% ~ 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation to prospectively design MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated high levels of mutant allele homogeneity. MMEJ-based DNA repair at these target loci successfully generated F0 mutant zebrafish embryos and larvae that faithfully recapitulated previously reported, recessive, loss-of-function phenotypes. We also tested the generalizability of our approach in cultured human cells. Finally, we provide a novel algorithm, MENTHU (http://genesculpt.org/menthu/), for improved and facile prediction of candidate MMEJ loci. We believe that this MMEJ-centric approach will have a broader impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an endogenous target will have important implications for gene therapy applications in human genetic diseases.

Janus kinase 2 (Jak2) transduces signals from hematopoietic cytokines, and a gainof-function mutation (Jak2 617V>F ) is associated with myeloproliferative diseases, particularly polycythemia vera. In this study, we examined the role of jak2a in zebrafish embryos in knock-down and overexpression studies using morpholinos (

Transcription activator-like effector nucleases (TALENs) enable genome engineering in cell culture and many organisms. Recently, the GoldyTALEN scaffold was shown to readily introduce mutations in zebrafish (Danio rerio) and livestock through non-homologous end joining (NHEJ) and homology-directed repair (HDR). To deploy the GoldyTALEN system for high-throughput mutagenesis in model organisms, a simple design with high efficacy is desirable. We tested the in vivo efficacy of a simplified 15-RVD GoldyTALEN design (spacer between 13–20 bp and T nucleotide preceding each TALEN binding site) in zebrafish. All 14 tested TALEN pairs (100%) introduced small insertions and deletions at somatic efficacy ranging from 24 to 86%, and mutations were inheritable at high frequencies (18–100%). By co-injecting two GoldyTALEN pairs, inheritable deletions of a large genomic fragment up to 18 kb were successfully introduced at two different loci. In conclusion, these high efficiency 15-RVD GoldyTALENs are useful for high-throughput mutagenesis in diverse application including hypothesis testing from basic science to precision medicine.

Transcription activator-like effectors (TALEs) are extremely effective, single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high-precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints toward the targeted genome than other custom nuclease systems. One bottleneck of broader TALE use is reagent accessibility. For example, one commonly deployed method uses a multitube, 5-day assembly protocol. Here we describe FusX, a streamlined Golden Gate TALE assembly system that (1) is backward compatible with popular TALE backbones, (2) is functionalized as a single-tube 3-day TALE assembly process, (3) requires only commonly used basic molecular biology reagents, and (4) is cost-effective. More than 100 TALEN pairs have been successfully assembled using FusX, and 27 pairs were quantitatively tested in zebrafish, with each showing high somatic and germline activity. Furthermore, this assembly system is flexible and is compatible with standard molecular biology laboratory tools, but can be scaled with automated laboratory support. To demonstrate, we use a highly accessible and commercially available liquid-handling robot to rapidly and accurately assemble TALEs using the FusX TALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications.