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Genetic Engineering Publications - GEG Tech top picks
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Two independent groups have now developed a new class of antimicrobials that act on specific bacterial populations, while leaving others unharmed. These new antimicrobials are based on the Streptococcus pyogenes type II CRISPR gene-editing system, which directs the Cas9 nuclease to cleave genomic target sites that can be specified in CRISPR guide RNAs (crRNAs). Lu and colleagues targeted enterobacterial genes encoding β-lactamase enzymes that conferred extended-spectrum or pan–β-lactam antibiotic resistance, whereas Bikard, Marraffini and colleagues studied the specific elimination of kanamycin-resistant or MRSA cells. Both groups showed that transforming bacteria with plasmids bearing Cas9 and crRNAs that targeted specific antibiotic-resistance factors was able to promote killing of the intended bacterial populations without affecting cells that were not carrying the targeted sequences. Lu and colleagues also demonstrated that Cas9–crRNA modules could be introduced into target bacterial cells through conjugation with engineered donor bacteria containing mobilizable plasmids or by infection with M13 phagemids. The latter approach was used to modulate the composition of a complex microbial community in vitro and was also efficient in treating Escherichia coli O157:H7 infection in an insect larva model. Bikard, Marraffini and colleagues used a phagemid-based approach to target kanamycin-resistant S. aureus mixed with kanamycin-sensitive bacteria and found that the nontargeted cells outcompeted any residual targeted cells for growth. The group also showed that a single crRNA construct could successfully be programmed against two separate virulence plasmids in an MRSA strain. Phagemid treatment of antibiotic-sensitive S. aureus could immunize the cells against the transfer of antibiotic-resistance genes from infection with phage grown on the MRSA strain. Selective targeting of kanamycin-resistant bacteria was also demonstrated in a mouse skin colonization model. Notably, both groups found that Cas9-targeted escapees that arose after treatment were due to defects in the CRISPR constructs rather than to host-adaptive mutations that created resistance to the new drug, thus supporting the concept of CRISPR-based treatments as an alternative to traditional drug therapies. 
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The scientists demonstrate a possible new mechanism by which HDACi influence tumorigenesis and apoptosis via downregulation of miR-125a-5p expression. This study provides clinical implications in cancer chemotherapy using HDACi.
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The authors describe a linear amplification–mediated modification of a previously published high-throughput, genome-wide, translocation sequencing (HTGTS) method that robustly detects DNA double-stranded breaks (DSBs) generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs. HTGTS with different Cas9:sgRNA or TALEN nucleases revealed off-target hotspot numbers for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases more than tenfold. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-target cleavage genome-wide.
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In this study, the scientists report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led not to efficient mutagenesis in T cells. However, their results results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy.
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The authors describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs.
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The first synthetic molecules that have both the targeting and response functions of antibodies have been crafted by scientists. The new molecules -- synthetic antibody mimics -- attach themselves simultaneously to disease cells and disease-fighting cells. The result is a highly targeted immune response, similar to the action of natural human antibodies.
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Roland Rad and colleagues report development of a new conditional piggyBac transposition system for performing insertional mutagenesis screens in mice. They apply the system to identify new oncogenic driver pathways for pancreatic cancer.
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Moisan, Cowan and colleagues perform a small-molecule screen to identify compounds that promote white-to-brown adipocyte conversion in vitro. They report that two inhibitors of the JAK–STAT signalling pathway stimulate browning of human adipocytes.
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This study describe a screening platform for the identification of small molecules capable of promoting a white-to-brown metabolic conversion in human adipocytes. The authors highlighted a previously unknown role for the JAK–STAT pathway in the control of adipocyte function and establish a platform to identify compounds for the treatment of obesity.
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The scientists describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. In this way, they demonstrated multiplexed activation of ten genes simultaneously, and the upregulation of long intergenic non-coding RNA (lincRNA) transcripts.
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In this Opinion article, scientists present a general scenario for the origin of adaptive immunity from mobile elements and innate immune systems.
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In this study, the authors investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both non-homologous end joining (NHEJ)-mediated gene disruption and homologous donor-based precise genome editing (HR). In these two contexts, they observed a higher efficency of the CRISPR/Cas9 system. In the first case (NHEJ), for three loci tested, Cas9-gRNAs induced between 10-100 fold more indels than did TALENs in human iPSCs, reaching the level of 0.7% to 2.5% mutation rates. About the second case (HR),the largest difference was observed at the AAVS1 site targeting where the Cas9-gRNA showed about 2-fold advantage over TALENs. geg-tech.com
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The authors combine whole-genome sequencing and deep-targeted sequencing to characterise the off-target effects of CRISPR/Cas9 system which targets Tafazzin gene. Based on in silico analysis, they estimate a likelihood of single-nucleotide variant creating off-target sites in a human genome to be ~1.5–8.5%, depending on the genome and site-selection method. geg-tech.com
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The authors review the fast developing technology of targeted genome engineering using site specific programmable nucleases zinc finger nucleases (ZFNs), transcription activator like nucleases (TALENs) and cluster regulatory interspaced short palindromic repeat/CRISPR associated proteins (CRISPR/Cas) based RNA-guided DNA endonucleases (RGENs) and their different characteristics including pros and cons of genome modifications by these nucleases. They have further discussed different types of delivery methods to induce gene editing, novel development in genetic engineering other than nucleases and future prospects.
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The scientists employed fibroblasts derived from a patient with Fanconi anemia as a model to test the ability of the clustered regularly interspaced short palindromic repeats/Cas9 nuclease system to mediate gene correction. They show that the Cas9 nuclease and nickase each resulted in gene correction, but the nickase, due to its ability to preferentially mediate homology-directed repair, resulted in a higher frequency of corrected clonal isolates.
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The authors introduce a methodology to control the copies and residence time of a gene product delivered in host human cells but also selectively disrupt fragments of the delivery vehicle.
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Injection of preneoplastic human mammary epithelial cells into the mammary ducts of immunodeficient mice leads to replacement of the murine luminal layer with morphologically normal human cells. Genetic manipulation of the injected cells by lentiviral vectors makes it possible to study defined steps in the transformation of human mammary epithelial cells in a more physiological environment than has hitherto been possible.
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The scientists demonstrate that, in a multifocal ovarian cancer model, polymer-delivered T cells trigger regression, whereas injected tumor-reactive lymphocytes have curative effect.
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The authors used CRISPR/Cas9 to correct β-Thal iPSCs, gene-corrected cells exhibit normal karyotypes and fully pluripotency as hES, showed no off-targeting effects. Their results shown that the gene-corrected β-Thal iPS cell lines restored HBB expression and reduced reactive oxygen species (ROS) production. www;geg-tech.com
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Giuseppe Testa and colleagues report the generation and transcriptional characterization of patient-derived induced pluripotent stem cells (iPSCs) with copy number variants at 7q11.23, which cause syndromes including neurocognitive phenotypes. They find that the dosage of the transcription factor gene GTF2I accounts for 10-20% of the transcriptional dysregulation observed in these cells.
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The authors compare Sendai-viral (SeV), episomal (Epi) and mRNA transfection mRNA methods using a number of criteria.
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The authors demonstrated that high-aspect-ratio, mesoporous silica rods (MSRs) injected with a needle spontaneously assemble in vivo to form macroporous structures that provide a 3D cellular microenvironment for host immune cells. In mice, substantial numbers of dendritic cells are recruited to the pores between the scaffold rods. These findings suggest that injectable MSRs may serve as a multifunctional vaccine platform to modulate host immune cell function and provoke adaptive immune responses.
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PLOS ONE: an inclusive, peer-reviewed, open-access resource from the PUBLIC LIBRARY OF SCIENCE. Reports of well-performed scientific studies from all disciplines freely available to the whole world.
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Using a CRISPR genome editing strategy the authors deleted a super-enhancer (SE) around the Sox2 gene in mouse embryonic stem cells (ESCs). The analysis of transcriptional defects showed that the SE is responsible for over 90% of Sox2 expression, suggesting that the functional significance of this SE is to maintain the pluripotency transcription program in mouse ESCs.
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PLOS ONE: an inclusive, peer-reviewed, open-access resource from the PUBLIC LIBRARY OF SCIENCE. Reports of well-performed scientific studies from all disciplines freely available to the whole world.
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The authors describe the generation of PrP knockout clones from epithelial NMuMG cell line using CRISPR-Cas9 knockout technology. The quantitative global proteome analysis demonstrates that ~120 proteins were shown to reproducibly correlate with the presence or absence of PrP, with most of these proteins belonging to extracellular components, cell junctions or the cytoskeleton.
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This review examines current status of genome editing in mammalian embryonic stem cells and zygotes and discuss potential issues in the international regulatory landscape regarding human germline gene modification. geg-tech.com
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A new study reports an extension of the CRISPR (clustered regularly interspaced short palindromic repeat) technology that enables the simultaneous, tunable and directional control of expression of multiple genes. At its most basic, this method allows switching on one set of genes while switching off another set to generate synthetic multigene transcriptional programmes that can be induced to, for example, 'rewire' cell fates or engineer metabolic pathways.
To achieve flexible locus-specific transcriptional programming at multiple genes simultaneously, the researchers took advantage of features inherent to RNA, such as its modularity and programmability: RNA has previously been shown to form scaffolds that link DNA binding through base pairing to protein recruitment and the assembly of functional protein complexes.
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