Transcription Activator-like Effector Nucleases Technology Research Articles

Targeted Mutagenesis in the Malaria Mosquito Using TALE Nucleases

Anopheles gambiae, the main mosquito vector of human malaria, is a challenging organism to manipulate genetically. As a consequence, reverse genetics studies in this disease vector have been largely limited to RNA interference experiments. Here, we report the targeted disruption of the immunity gene TEP1 using transgenic expression of Transcription-Activator Like Effector Nucleases (TALENs), and the isolation of several TEP1 mutant A. gambiae lines. These mutations inhibited protein production and rendered TEP1 mutants hypersusceptible to Plasmodium berghei. The TALEN technology opens up new avenues for genetic analysis in this disease vector and may offer novel biotechnology-based approaches for malaria control.

Efficient drug screening and gene correction for treating liver disease using patient-specific stem cells

Patient-specific induced pluripotent stem cells (iPSCs) represent a potential source for developing novel drug and cell therapies. Although increasing numbers of disease-specific iPSCs have been generated, there has been limited progress in iPSC-based drug screening/discovery for liver diseases, and the low gene-targeting efficiency in human iPSCs warrants further improvement. Using iPSC lines from patients with alpha-1 antitrypsin (AAT) deficiency, for which there is currently no drug or gene therapy available, we established a platform to discover new drug candidates and correct disease-causing mutation with a high efficiency. A high-throughput format screening assay, based on our hepatic differentiation protocol, was implemented to facilitate automated quantification of cellular AAT accumulation using a 96-well immunofluorescence reader. To expedite the eventual application of lead compounds to patients, we conducted drug screening utilizing our established library of clinical compounds (the Johns Hopkins Drug Library) with extensive safety profiles. Through a blind large-scale drug screening, five clinical drugs were identified to reduce AAT accumulation in diverse patient iPSC-derived hepatocyte-like cells. In addition, using the recently developed transcription activator-like effector nuclease technology, we achieved high gene-targeting efficiency in AAT-deficiency patient iPSCs with 25%-33% of the clones demonstrating simultaneous targeting at both diseased alleles. The hepatocyte-like cells derived from the gene-corrected iPSCs were functional without the mutant AAT accumulation. This highly efficient and cost-effective targeting technology will broadly benefit both basic and translational applications. Our results demonstrated the feasibility of effective large-scale drug screening using an iPSC-based disease model and highly robust gene targeting in human iPSCs, both of which are critical for translating the iPSC technology into novel therapies for untreatable diseases.

Transcription activator‐like effector nucleases (TALENs): A highly efficient and versatile tool for genome editing

Transcription activator-like effector (TALE) nucleases (TALENs) have recently emerged as a revolutionary genome editing tool in many different organisms and cell types. The site-specific chromosomal double-strand breaks introduced by TALENs significantly increase the efficiency of genomic modification. The modular nature of the TALE central repeat domains enables researchers to tailor DNA recognition specificity with ease and target essentially any desired DNA sequence. Here, we comprehensively review the development of TALEN technology in terms of scaffold optimization, DNA recognition, and repeat array assembly. In addition, we provide some perspectives on the future development of this technology.

Targeted genome engineering in human induced pluripotent stem cells by penetrating TALENs.

BackgroundZinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research. However, the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases. A new delivery method that can improve the utility of these nucleases is needed.ResultsIn this study, we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery. Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN, respectively. However, TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture. Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine (C-C motif) receptor 5 (CCR5, a co-receptor for HIV-1 entry into cells). Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency. A 5% modification rate was observed in human induced pluripotent stem cells (hiPSCs) treated with TAT-TALEN as measured by the Surveyor assay.ConclusionsTAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells. This new technique may advance the clinical application of TALEN technology.Electronic supplementary materialThe online version of this article (doi:10.1186/2045-9769-2-5) contains supplementary material, which is available to authorized users.

Talens-Mediated DLEU1 Gene Silencing in Burkitt Lymphoma (BL): Implication of Lncrna DLEU1 Gene As a Potential Tumor Suppressor Gene in BL.

AbstractAbstract 2403 Background:Pediatric Burkitt Lymphoma (BL) is the most common histological subtype of Non Hodgkin Lymphoma (NHL) in children and adolescents (Cairo et al., Blood, 2007; Miles/Cairo, BJHaem., 2012). We previously identified in a subset analysis that children with BL and a 13q deletion, particularly 13q14.3, had significantly poorer outcome and inferior overall survival (OS) despite aggressive short, intensive multiagent chemotherapy (Poirel/Cairo et al., Leukemia, 2009; Nelson/Cairo/Sanger et al., BJHaem., 2009). Deleted in Lymphocytic Leukemia 1 (DLEU1) is a long non codign RNA gene with the BL classifier genes as reported by Dave et al (NEJM. 2006) on chromosome 13q14.3 region and the expression levels of RASSF1, TUBB2C and UBR1 were significantly higher in BL than in diffuse large B-cell lymphoma (DLBCL) (Day/Cairo et al., AACR, 2008). Sequence-specific Zinc Finger Nucleases (ZFN) and Transcription Activator-Like Effector Nucleases (TALENs) technologies have been developed for precision targeted genome editing in in vitro experiments with high efficiency and TALEN technology has been applied in a variety of organisms and stem cells for new experimental and therapeutic tools (Carlson et al., Mol Ther., 2012). Objectives:We hypothesize that 1) TALEN technology is suitable for the modification of and silencing of DLEU1 locus and 2) DLEU1 may have function as a tumor suppressor gene and therefore examined whether the loss of DLEU1 by TALEN-mediated DLEU1 knockdown Raji BL cells result in change in proliferation and programmed cell death. Methods:TALENs were constructed based on REAL (restriction enzyme and ligation) assembly methods (Sander et al., Nat. Biotech, 2011) for DLEU1 gene modification. DLEU1 TALEN pairs or combinations were transfected into 293T and Raji cells using lipofectamin reagent (Invitrogen) and Amaxa nucleofection kit, respectively. Genomic DNA and total RNA were extracted using genomic DNA extraction kit (Promega) and Trizol reagent (Invitrogen). Surveyor mutation detection assay (Transgenomic) was applied for TALENs validities and confirmed by sequencing analysis. Quantitative RT-PCR was performed by CFX96 Real-time system (Bio-rad) using qScript™ cDNA Synthesis Kit (Quantas) and SsoFast™ EvaGreen® Supermix (Bio-rad). Transfected cells were re-plated (1×104) into 48 well plates at 24hours post transfection and then counted every 24hrs for cell proliferation assay. For spontaneous apoptosis assay, DLEU1 TALENs transfected Raji cells were re-plated into 96 well plates at 24hours post-transfection and measured caspase 3/7 activities (Promega) using Clarity Luminescence microplate reader (Biotek) and statistical significance was determined by a one or two-tailed Student t-test. Results:Three pairs of functional DLEU1 TALENs (T1, T2, and T3) targeting endogenous DLEU1 gene generated and confirmed their validities with gene modification caused by non-homologous end joining (NHEJ) in TALENs mediated 293T [Figure 1]. Forty eight hours after transiently transfection in Raji cells, combined DLEU1 TALENs, T1/T2 (T12), T1/T3 (T13) and T2/T3 (T23) mediated Raji cells were dramatically reduced expression of DLEU1 mRNA (about 82–93% reduction) compared to untransfected Raji cells [92% reduction (p<0.002), 93% (p<0.0006), and 92% (p<0.0003)], respectively, and compared to empty vector as a control [82% reduction (p<0.05), 87% (p<0.0001), and 82% (p<0.001, respectively)]. T12 and T13-mediated DLEU1 knockdown Raji cells showed significantly an increase of cell proliferation [1.2-fold (p<0.02) and 1.5-fold (p<0.04) at day 5, respectively]. In spontaneous apoptotic assays, T13 mediated Raji showed significantly 20% (p<0.05) of reduction of caspase 3/7 activities. In comparison of mRNA expression of DLEU1 network genes in T13 mediated Raji cells, there were no significant differences in mRNA expression of c-myc compared to control, however, there was significantly reduced expression of RASSF1 and UBR1 mRNA (<7.7-fold, p<0.005 and <11.1-fold, p<0.0005), respectively, and significantly increased expression of TUBB2C mRNA (>7.5-fold, p<0.005). Conclusions:We demonstrate that 1) DLEU1-TALENs are useful tools for the modification of endogenous DLEU1 gene expression, 2) DLEU1 may have as a tumor suppressor gene in BL, and 3) DLEU1 TALEN mediated gene knockdown resulted in inhibition of BL apoptosis and increase in cell proliferation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs)

Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%–76.8% compared to 1.1%–3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

FLASH assembly of TALENs for high-throughput genome editing

Engineered transcription activator-like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) platform, a rapid and cost-effective method we developed to enable large-scale assembly of TALENs. We tested 48 FLASH-assembled TALEN pairs in a human cell-based EGFP reporter system and found that all 48 possessed efficient gene modification activities. We also used FLASH to assemble TALENs for 96 endogenous human genes implicated in cancer and/or epigenetic regulation and found that 84 pairs were able to efficiently introduce targeted alterations. Our results establish the robustness of TALEN technology and demonstrate that FLASH facilitates high-throughput genome editing at a scale not currently possible with engineered zinc-finger nucleases or meganucleases.

Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy.