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Scooped by
dromius
November 28, 2015 4:41 AM
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Tobita et al, 2015 In the recent decades, human genome engineering has been one of the major interesting research subjects, essentially because it raises new possibilities for personalized medicine and biotechnologies. With the development of engineered nucleases such as the Zinc Finger Nucleases (ZFNs), the Transcription activator-like effector nucleases (TALENs) and more recently the Clustered Regularly Interspaced short Palindromic Repeats (CRISPR), the field of human genome edition has evolved very rapidly. Every new genetic tool is broadening the scope of applications on human tissues, even before we can completely master each of these tools. In this review, we will present the recent advances regarding human genome edition tools, we will discuss the numerous implications they have in research and medicine, and we will mention the limits and concerns about such technologies
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Scooped by
dromius
November 19, 2015 3:45 AM
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(via T. Schreiber, thx) Miyamoto et al, 2015 Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus , a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, f
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Scooped by
dromius
November 14, 2015 10:02 AM
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Mesarich et al 2015 Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms.
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Scooped by
dromius
November 10, 2015 4:19 AM
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Silva et al, 2015 The establishment of Left-Right asymmetry in vertebrates is dependent on the sensory and motile functions of cilia during embryogenesis. Mutations in CCDC11 disrupt l-R asymmetry and cause congenital heart disease in humans, yet the molecular and cellular functions of the protein remain unknown. Here, we demonstrate that Ccdc11 is a novel component of centriolar satellites, cytoplasmic granules that serve as recruitment sites for proteins destined for the centrosome and cilium. Ccdc11 interacts with core components of satellites, and its loss disrupts the subcellular organization of satellite proteins and perturbs primary cilium assembly. Ccdc11 colocalizes with satellite proteins in human multiciliated tracheal epithelia, and its loss inhibits motile ciliogenesis. Similarly, depletion of CCDC11 in Xenopus embryos causes defective assembly and motility of cilia in multiciliated epidermal cells. To determine the role of CCDC11 during vertebrate development, we generated mutant alleles in zebrafish. Loss of CCDC11 leads to defective ciliogenesis in the pronephros and within the Kupffer's vesicle, and results in aberrant l-R axis determination. Our results highlight a critical role for Ccdc11 in the assembly and function of motile cilia, and implicate centriolar satellite-associated proteins as a new class of proteins in the pathology of l-R patterning and congenital heart disease.
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Scooped by
dromius
November 10, 2015 4:16 AM
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Garate et al, 2015 Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.
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Scooped by
dromius
November 7, 2015 12:53 PM
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Geissler et al, 2015 The major obstacle to cure infections with human immunodeficiency virus (HIV-1) is integrated proviral genomes, which are not eliminated by antiretroviral therapies (ART). Treatment approaches with latency-reversing agents (LRAs) aim at inducing provirus expression to tag latently-infected cells for clearance through viral cytopathic effects or cytotoxic T cell (CTL) responses. However, the currently tested LRAs reveal evident drawbacks as gene expression is globally induced and viral outgrowth is insecure. Here, we present transcription activator-like effector (TALE) proteins as potent tools to activate HIV-1 specifically. The large variety of circulating HIV-1 strains and, accordingly, integrated proviruses was addressed by the programmable DNA-specificity of TALEs. Using customized engineered TALEs, a substantial transcription activation and viral outgrowth was achieved with cells obtained from different HIV-1 patients. Our data suggest that TALEs may be useful tools in future strategies aimed at removing HIV-1 reservoirs.
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Scooped by
dromius
November 6, 2015 3:55 AM
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TALEN mediated gene-editing technique has been used to eliminate an infant’s aggressive cancer, a small but significant step in the quest to treat deadly diseases by altering the human genome.
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Scooped by
dromius
November 6, 2015 2:57 AM
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(via T. Lahaye, thx)
Čermák et al, 2015 The use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA.
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Scooped by
dromius
November 1, 2015 4:12 PM
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Vierstra et al, 2015 Regulatory regions harbor multiple transcription factor (TF) recognition sites; however, the contribution of individual sites to regulatory function remains challenging to define. We describe an approach that exploits the error-prone nature of genome editing–induced double-strand break repair to map functional elements within regulatory DNA at nucleotide resolution. We demonstrate the approach on a human erythroid enhancer, revealing single TF recognition sites that gate the majority of downstream regulatory function.
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Scooped by
dromius
October 28, 2015 5:02 AM
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(via T. Schreiber, thx) Novodvorsky et al, 2015 Using Transcription Activator-Like Effector Nucleases (TALEN) we generated a klf2a mutant (klf2ash317) with a 14bp deletion leading to a premature stop codon in exon 2. Western blotting confirmed loss of wild type Klf2a protein and the presence of a truncated protein in klf2ash317 mutants. Homozygous klf2ash317 mutants exhibit no defects in vascular patterning, survive to adulthood and are fertile, without displaying previously described morphant phenotypes such as high-output cardiac failure, reduced haematopoetic stem cell (HSC) development or impaired formation of the 5th accessory aortic arch. Homozygous klf2ash317 mutation did not reduce angiogenesis in zebrafish with homozygous mutations in von Hippel Lindau (vhl), a form of angiogenesis that is dependent on blood flow. We examined expression of three klf family members in wildtype and klf2ash317 zebrafish. We detected vascular expression of klf2b (but not klf4a or biklf/klf4b/klf17) in wildtypes but found no differences in expression that might account for the lack of phenotype in klf2ash317 mutants. klf2b morpholino knockdown did not affect heart rate or impair formation of the 5th accessory aortic arch in either wildtypes or klf2ash317 mutants.
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Scooped by
dromius
October 27, 2015 4:47 AM
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(via T. Lahaye, thx) Shiomi et al, 2015 The insect neuropeptide family FXPRLa, which carries the Phe-Xaa-Pro-Arg-Leu-NH2 sequence at the C-terminus, is involved in many physiological processes. Although ligand–receptor interactions in FXPRLa signaling have been examined using in vitro assays, the correlation between these interactions and in vivo physiological function is unclear. Diapause in the silkworm, Bombyx mori, is thought to be elicited by diapause hormone (DH, an FXPRLa) signaling, which consists of interactions between DH and DH receptor (DHR). Here, we performed transcription activator-like effector nuclease (TALEN)-based mutagenesis of the Bombyx DH-PBAN and DHR genes and isolated the null mutants of these genes in a bivoltine strain. All mutant silkworms were fully viable and showed no abnormalities in the developmental timing of ecdysis or metamorphosis. However, female adults oviposited non-diapause eggs despite diapause-inducing temperature and photoperiod conditions. Therefore, we conclude that DH signaling is essential for diapause induction and consists of highly sensitive and specific interactions between DH and DHR selected during ligand–receptor coevolution in Bombyx mori.
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Scooped by
dromius
October 20, 2015 5:38 AM
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(via t. Lahaye, thx) de Lange & Wolf et al, 2015 Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats.
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Scooped by
dromius
October 15, 2015 3:02 AM
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Fusion proteins comprising a DNA binding domain, e.g., a TAL effector repeat array (TALE) or zinc finger array, and a catalytic domain comprising a sequence that catalyzes histone demethylation, and m
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Scooped by
dromius
November 24, 2015 2:53 AM
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Du et al, 2015 Gene targeting (GT) is of great significance for advancing basic plant research and crop improvement. Both TALENs (transcription activator-like effectors nucleases) and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) systems have been developed for genome editing in eukaryotes, including crop plants. In this work, we present the comparative analysis of these two technologies for two soybean genome editing targets, GmPDS11 and GmPDS18. We found GT in soybean hairy roots with a single targeting efficiency range of 17.5% to 21.1% by TALENs, 11.7% to 18.1% by CRISPR/Cas9 using the AtU6-26promoter, and 43.4% to 48.1% by CRISPR/Cas9 using the GmU6-16g-1promoter, suggesting that the CRISPR/Cas9 using the GmU6-16g-1promoter is probably a much more efficient tool compared to the other technologies. Similarly, our double mutation GT efficiency experiment with these three technologies displayed a targeting efficiency of 6.25% by TALENs, 12.5% by CRISPR/Cas9 using the AtU6-26promoter, and 43.4% to 48.1% by CRISPR/Cas9 using the GmU6-16g-1promoter, suggesting that CRISPR/Cas9 is still a better choice for simultaneous editing of multiple homoeoalleles. Furthermore, we observed albino and dwarf buds (PDS knock-out) by soybean transformation in cotyledon nodes. Our results demonstrated that both TALENs and CRISPR/Cas9 systems are powerful tools for soybean genome editing.
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Scooped by
dromius
November 18, 2015 9:01 AM
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Cohn et al, 2015 Xanthomonas axonopodis pv. manihotis (Xam) employs transcription activator like (TAL) effectors to promote bacterial growth and symptom formation during infection of cassava. TAL effectors are secreted via the bacterial type III secretion system into plant cells where they are directed to the nucleus, bind DNA in plant promoters, and activate the expression of downstream genes. The DNA binding activity of TAL effectors is carried out by a central domain which contains a series of repeat variable diresidues (RVDs) that dictate the sequence of bound nucleotides. TAL14Xam668 promotes virulence in Xam strain Xam668 and has been shown to activate multiple cassava genes. In this study we used RNA-sequencing to identify the full target repertoire of TAL14Xam668 in cassava which includes over 50 genes. A subset of highly upregulated genes were tested for activation by TAL14CIO151 from Xam strain CIO151. Although TAL14CIO151 and TAL14Xam668 differ by only a single RVD they display differential activation of gene targets. TAL14CIO151 complements the TAL14Xam668 mutant defect, implying that shared target genes are important for TAL14Xam668-mediated disease susceptibility. Complementation with closely related TAL effectors is a novel approach to narrowing down biologically relevant susceptibility genes of TAL effectors with multiple targets. This study provides an example of how TAL effector target activation by two strains within a single species of Xanthomonas can be dramatically affected by a small change in RVD-nucleotide affinity at a single site and reflects the parameters of RVD-nucleotide interaction determined using designer TAL effectors in transient systems. This article is protected by copyright. All rights reserved.
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Scooped by
dromius
November 12, 2015 4:11 AM
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(via T. Lahaye, thx) Chiang et al, 2015 Although myostatin, a suppressor of skeletal muscle development and growth, has been well studied in mammals, its function in fish remains unclear. In this study, we used a popular genome editing tool with high efficiency and target specificity (TALENs; transcription activator-like effector nucleases) to mutate the genome sequence of myostatin (MSTN) in medaka (Oryzias latipes). After the TALEN pair targeting OlMyostatin was injected into fertilized medaka eggs, mutant G0 fish carrying different TALENs-induced frameshifts in the OlMSTN coding sequence were mated together in order to transmit the mutant sequences to the F1 generation. Two F1 mutants with frameshifted myostatin alleles were then mated to produce the F2 generation, and these F2 OlMSTN null (MSTN-/-) medaka were evaluated for growth performance. The F2 fish showed significantly increased body length and weight compared to the wild type fish at the juvenile and post-juvenile stages. At the post-juvenile stage, the average body weight of the MSTN-/- medaka was ∼25% greater than the wild type. However, we also found that when the F3 generation were challenged with red spotted grouper nervous necrosis virus (RGNNV), the expression levels of the interferon-stimulated genes were lower than in the wild type, and the virus copy number was maintained at a high level. We therefore conclude that although the MSTN-/- medaka had a larger phenotype, their immune system appeared to be at least partially suppressed or undeveloped.
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Scooped by
dromius
November 10, 2015 4:17 AM
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Van Hove et al 2015 Transcription is a complex and dynamic process representing the first step in gene expression that can be readily controlled through current tools in molecular biology. Elucidating and subsequently controlling transcriptional processes in various prokaryotic and eukaryotic organisms have been a key element in translational research, yielding a variety of new opportunities for scientists and engineers. This chapter aims to give an overview of how the fields of molecular and synthetic biology have contributed both historically and presently to the state of the art in transcriptional engineering. The described tools and techniques, as well as the emerging genetic circuit engineering discipline, open the door to new advances in the fields of medical and industrial biotechnology.
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Scooped by
dromius
November 10, 2015 4:13 AM
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Weninger et al, 2015 The advent of potent genome engineering techniques and efficient DNA assembly methods has greatly expanded our capacities to modify genomes and have fueled metabolic engineering and synthetic biology endeavours. Here we provide an overview on genome engineering methods ranging from non-programmable tools such as site-specific recombinases to highly specific, programmable strategies as CRISPR–Cas9. Advantages and limitations of the methods for the generation of custom-designed organisms are compared. Certain methods such as group II introns and recombineering are limited to prokaryotes, whereas zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs) and CRISPR–Cas9 are universally applicable for all domains of life. We summarize also popular DNA assembly methods, suitable for generating multigene pathways for metabolic engineering and artificial constructs for synthetic biology or entire genomes.
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Scooped by
dromius
November 7, 2015 12:51 PM
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Booher et al, 2015 Pathogen-injected, direct transcriptional activators of host genes, TAL (transcription activator-like) effectors play determinative roles in plant diseases caused by Xanthomonas spp. A large domain of nearly identical, 33–35 aa repeats in each protein mediates DNA recognition. This modularity makes TAL effectors customizable and thus important also in biotechnology. However, the repeats render TAL effector (tal) genes nearly impossible to assemble using next-generation, short reads. Here, we demonstrate that long-read, single molecule real-time (SMRT) sequencing solves this problem. Taking an ensemble approach to first generate local, tal gene contigs, we correctly assembled de novo the genomes of two strains of the rice pathogen X. oryzae completed previously using the Sanger method and even identified errors in those references. Sequencing two more strains revealed a dynamic genome structure and a striking plasticity in tal gene content. Our results pave the way for population-level studies to inform resistance breeding, improve biotechnology and probe TAL effector evolution.
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Scooped by
dromius
November 6, 2015 3:50 AM
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Orlando de Lange, 2015 In the introduction of this dissertation, which explores TALE biology, a particular focus will be placed on the DNA binding properties of TALEs and how this can be put to use in TALE technology. After this the RipTALs, Bats and MOrTLs are each introduced, explaining what is known about their provenance and sequence features. The aims of my doctoral work are then listed and expounded in turn. The proximal goal of my doctoral work was to carry out a comparative molecular characterization of each group of non-TALE TALE-likes. In doing so we hoped to gain insights into the principles of TALE-like DNA-binding properties, evolutionary history of the different groups and their potential uses in biotechnology. In the case of the RipTALs this work should begin to unravel the role these proteins play in bacterial wilt disease, as a means to fight this devastating pathogen. The articles I have worked on covering the molecular characterizations of RipTALs, Bats and MOrTLs are then presented in turn. Working together with others I was able to show that repeats from each group of TALE-likes mediate sequence specific DNA binding, revealing a conserved code in each case. This code links position 13 of any TALE-like repeat to a specific DNA base preference in a reliable fashion. I will argue that the TALE-likes represent a fascinating case of conserved structure and function in a diverse sequence space. In addition the TALEs and RipTALs may simply represent one face of the TALE-likes, a protein family mediating as yet unknown biological roles as bacterial DNA binding proteins.
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Scooped by
dromius
November 5, 2015 3:49 AM
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(via T. Schreiber, thx) Ding et al, 2015 The inner mitochondrial membrane (IMM) invaginates to form cristae and the maintenance of cristae depends on the mitochondrial contact site (MICOS) complex. Mitofilin and CHCHD6, which physically interact, are two components of the MICOS. In this study, we performed immunoprecipitation experiments with Mitofilin and CHCHD6 antibodies and identified a complex containing Mitofilin, Sam50, and CHCHD 3 and 6. Using transcription activator-like effector nucleases (TALENs), we generated knockdown/knockout clones of Mitofilin and CHCHD6. Transmission electron microscopy (TEM) revealed that vesicle-like cristae morphology appeared in cell lines lacking Mitofilin, and mitochondria exhibited lower cristae density in CHCHD6-knockout cells. Immunoblot analysis showed that knockdown of Mitofilin, but not knockout of CHCHD6, affected their binding partners that control cristae morphology. We also demonstrated that Mitofilin and CHCHD6 directly interacted with Sam50. Additionally, we observed that Mitofilin-knockdown cells showed decreased mitochondrial membrane potential (ΔΨm) and intracellular ATP content, which were minimally affected in CHCHD6-knockout cells. Taken together, we conclude that the integrity of MICOS and its efficient interaction with Sam50 are indispensable for cristae organization, which is relevant to mitochondrial function.
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Scooped by
dromius
October 28, 2015 5:03 AM
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(via T. Lahaye and T. Schreiber, thx both) Menger et al, 2015 Cytomegalovirus (CMV) infection is responsible for substantial morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). T cell immunity is critical for control of CMV infection and correction of the immune deficiency induced by transplantation is now clinically achievable by the adoptive transfer of donor-derived CMV-specific T cells. It is notable, however, that most clinical studies of adoptive T cell therapy exclude patients with graft-versus-host disease (GvHD) receiving systemic corticosteroid therapy, which impairs cellular immunity. This group of patients remains the highest clinical risk group for recurrent and problematic infections. Here, we address this unmet clinical need by genetic disruption of the glucocorticoid receptor (GR) gene using electroporation of transcription activator-like effector nucleases (TALEN) mRNA. We demonstrate efficient inactivation of the GR gene without off-target activity in Streptamer®-selected CMV-specific CD8+ T cells (A2-NLV), conferring resistance to glucocorticoids. TALEN-modified CMV-specific T cells retained specific killing of target cells pulsed with the CMV peptide NLV in the presence of dexamethasone (DEX). Inactivation of the GR gene also conferred resistance to DEX in a xenogeneic-GvHD model in sub-lethally irradiated NOD-scid IL2rγnull (NSG) mice. This proof of concept provides the rationale for the development of clinical protocols for producing and administering high-purity genetically-engineered virus-specific T cells that are resistant to the suppressive effects of corticosteroids.
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Scooped by
dromius
October 27, 2015 4:54 AM
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Chandrasegaran & Carroll 2015 Genome engineering with programmable nucleases depends on cellular responses to a targeted double-strand break (DSB). The first truly targetable reagents were the zinc finger nucleases (ZFNs) that showed that arbitrary DNA sequences could be addressed for cleavage by protein engineering, ushering in the breakthrough in genome manipulation. ZFNs resulted from basic research on zinc finger proteins (ZFPs) and FokI restriction enzyme (which revealed a bipartite structure with a separable DNA-binding domain and a non-specific cleavage domain). Studies on the mechanism of cleavage by 3-finger ZFNs established that the preferred substrates were paired binding sites, which doubled the size of the target sequence recognition from 9 to 18 bp, long enough to specify a unique genomic locus in plant and mammalian cells. Soon afterwards, a ZFN-induced DSB was shown to stimulate homologous recombination (HR) in cells. Transcription activator-like effector nucleases (TALENs) that are based on bacterial TALEs fused to the FokI cleavage domain, expanded this capability. The fact that ZFNs and TALENs have been used for genome modification of more than 40 different organisms and cell types attests to the success of protein engineering. The most recent technology platform for delivering a targeted DSB to cellular genomes is that of the RNA-guided nucleases, which are based on the naturally occurring Type II prokaryotic CRISPR-Cas9 system. Unlike ZFNs and TALENs that use protein motifs for DNA sequence recognition, CRISPR-Cas9 depends on RNA-DNA recognition. The advantages of the CRISPR-Cas9 system - the ease of RNA design for new targets and the dependence on a single, constant Cas9 protein - have led to its wide adoption by research laboratories around the world. These technology platforms have equipped scientists with an unprecedented ability to modify cells and organisms almost at will, with wide-ranging implications across biology and medicine. However, these nucleases have also been shown to cut at off-target sites with mutagenic consequences. Therefore, issues like efficacy, specificity and delivery are likely to drive selection of reagents for particular purposes. Human therapeutic applications of these technologies will ultimately depend on risk vs. benefit analysis and informed consent.
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Scooped by
dromius
October 23, 2015 6:36 AM
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(via T. Schreiber, thx) Liu et al, 2015 Targeted nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However, realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids, the direct delivery of nuclease proteins to cells provides rapid action and fast turnover, leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression, purification and delivery of ZFN proteins, which are intrinsically cell-permeable; TALEN proteins, which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein, whose nucleofection into cells facilitates rapid induction of multiplexed modifications, are described, along with procedures for evaluating nuclease protein activity. Once they are constructed, nuclease proteins can be expressed and purified within 6 d, and they can be used to induce genomic modifications in human cells within 2 d.
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Scooped by
dromius
October 19, 2015 8:55 AM
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(via T. Lahaye, thx) Geissler et al, 2015 The major obstacle to cure infections with human immunodeficiency virus (HIV-1) is integrated proviral genomes, which are not eliminated by antiretroviral therapies (ART). Treatment approaches with latency-reversing agents (LRAs) aim at inducing provirus expression to tag latently-infected cells for clearance through viral cytopathic effects or cytotoxic T cell (CTL) responses. However, the currently tested LRAs reveal evident drawbacks as gene expression is globally induced and viral outgrowth is insecure. Here, we present transcription activator-like effector (TALE) proteins as potent tools to activate HIV-1 specifically. The large variety of circulating HIV-1 strains and, accordingly, integrated proviruses was addressed by the programmable DNA-specificity of TALEs. Using customized engineered TALEs, a substantial transcription activation and viral outgrowth was achieved with cells obtained from different HIV-1 patients. Our data suggest that TALEs may be useful tools in future strategies aimed at removing HIV-1 reservoirs.
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