Genetic Engineering - GEG Tech top picks
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DNA aptamers against FokI nuclease domain for genome editing applications

DNA aptamers against FokI nuclease domain for genome editing applications | Genetic Engineering - GEG Tech top picks | Scoop.it
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Here, the scientists report the development of DNA aptamers that bind to the target molecules, with high affinity and specificity to the FokI. DNA aptamers were selected in six rounds of systematic evolution of ligands by exponential enrichment. They find two aptamers which showed high binding affinity to FokI, resistance to nuclease activity itself and did not inhibit nuclease activity. These aptamers could be useful for genome editing applications such as controlled delivery of SSNs.

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Rapidly evolving homing CRISPR barcodes - Nature Methods 

Rapidly evolving homing CRISPR barcodes - Nature Methods  | Genetic Engineering - GEG Tech top picks | Scoop.it
Homing guide RNAs that target Cas9 to their own loci generate diverse barcodes that can trace the lineage of cells.
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In this study, the scientists present an approach for engineering evolving DNA barcodes in living cells. A homing guide RNA (hgRNA) scaffold directs the Cas9–hgRNA complex to the DNA locus of the hgRNA itself. This integrated approach will have wide-ranging applications, such as in deep lineage tracing, cellular barcoding, molecular recording, dissecting cancer biology, and connectome mapping.

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Applying CRISPR/Cas for genome engineering in plants: the best is yet to come

Applying CRISPR/Cas for genome engineering in plants: the best is yet to come | Genetic Engineering - GEG Tech top picks | Scoop.it
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Less than 5 years ago the CRISPR/Cas nuclease was first introduced into eukaryotes, shortly becoming the most efficient and widely used tool for genome engineering. For plants, efforts were centred on obtaining heritable changes in most transformable crop species by inducing mutations into open reading frames of interest, via non-homologous end joining.

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An open-hardware platform for optogenetics and photobiology

An open-hardware platform for optogenetics and photobiology | Genetic Engineering - GEG Tech top picks | Scoop.it
In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision.
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Here, the scientists engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day.

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Non-viral delivery of genome-editing nucleases for gene therapy

Non-viral delivery of genome-editing nucleases for gene therapy | Genetic Engineering - GEG Tech top picks | Scoop.it
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Herein, the authors summarize recent advances that have been made on non-viral delivery of genome-editing nucleases. In particular, we focus on non-viral delivery of Cas9/sgRNA ribonucleoproteins (RNPs) for genome editing. Additionally, the future direction for developing non-viral delivery of programmable nucleases for genome editing is discussed.

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From profiles to function in epigenomics : Nature Reviews Genetics : Nature Research

From profiles to function in epigenomics : Nature Reviews Genetics : Nature Research | Genetic Engineering - GEG Tech top picks | Scoop.it
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In this chapter, the authors discuss the current state of epigenomic profiling and how functional information can be indirectly inferred. They also present new approaches that promise definitive functional answers, which are collectively referred to as 'epigenome editing'. In particular, they explore CRISPR-based technologies for single-locus and multi-locus manipulation. Finally, they discuss which level of function can be achieved with each approach and introduce emerging strategies for high-throughput progression from profiles to function.

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Engineering cell signaling modulators from native protein–protein interactions

Engineering cell signaling modulators from native protein–protein interactions | Genetic Engineering - GEG Tech top picks | Scoop.it
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Here, the authors review the most recent progress on engineering natural protein–protein interactions for modulation of cell signaling.

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Scientists tissue-engineer human intestines and functioning nerves

Scientists tissue-engineer human intestines and functioning nerves | Genetic Engineering - GEG Tech top picks | Scoop.it
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Scientists report in Nature Medicine ("Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system") using human pluripotent stem cells to grow human intestinal tissues that have functioning nerves in a laboratory, and then using these to recreate and study a severe intestinal nerve disorder called Hirschsprung’s disease.

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Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA

Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA | Genetic Engineering - GEG Tech top picks | Scoop.it
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In this study, the authors report that multiple gRNAs linked with self-cleaving ribozymes and/or tRNA could be simultaneously expressed from a single U6 promoter to exert genome editing of dystrophin and myosin binding protein C3 in human and mouse cells.

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Genomics: RNA detection with C2c2 - Nature Methods 

Genomics: RNA detection with C2c2 - Nature Methods  | Genetic Engineering - GEG Tech top picks | Scoop.it
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The discovery of two distinct endonuclease activities in the C2c2 protein explains how template RNAs are processed in type VI CRISPR systems and enables the development of a sensitive RNA detection system.

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Contracting CAG/CTG repeats using the CRISPR-Cas9 nickase

Contracting CAG/CTG repeats using the CRISPR-Cas9 nickase | Genetic Engineering - GEG Tech top picks | Scoop.it
The expansion of trinucleotide repeats has been linked to several neurodegenerative disorders.
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CAG/CTG repeat expansions cause over 13 neurological diseases that remain without a cure. Because longer tracts cause more severe phenotypes, contracting them may provide a therapeutic avenue. In this study, the scientists find that inducing double-strand breaks within the repeat tract causes instability in both directions. In contrast, the CRISPR-Cas9 D10A nickase induces mainly contractions independently of single-strand break repair. They propose that DNA gaps lead to contractions and that the type of DNA damage present within the repeat tract dictates the levels and the direction of CAG repeat instability. This study paves the way towards deliberate induction of CAG/CTG repeat contractions in vivo.

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A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells

A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells | Genetic Engineering - GEG Tech top picks | Scoop.it
Hultquist et al. report a high-throughput platform for the efficient, multiplex editing
of host factors that control HIV infection in primary CD4+ T cells. Arrayed electroporation
of CRISPR/Cas9 ribonucleoproteins (RNPs) permits the rapid generation of isogenic
human cells with ablated candidate factors and identifies gene modifications that
provide viral resistance.
BigField GEG Tech's insight:

Here, the scientists adapted this methodology to a high-throughput platform for the efficient, arrayed editing of candidate host factors. CXCR4 or CCR5 knockout cells generated with this method are resistant to HIV infection in a tropism-dependent manner, whereas knockout of LEDGF or TNPO3 results in a tropism-independent reduction in infection. CRISPR/Cas9 RNPs can furthermore edit multiple genes simultaneously, enabling studies of interactions among multiple host and viral factors. Finally, in an arrayed screen of 45 genes associated with HIV integrase, they identified several candidate dependency/restriction factors, demonstrating the power of this approach as a discovery platform. This technology should accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection.

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Optogenetic activation of dopamine neurons in the ventral tegmental area induces reanimation from general anesthesia

Optogenetic activation of dopamine neurons in the ventral tegmental area induces reanimation from general anesthesia | Genetic Engineering - GEG Tech top picks | Scoop.it
National Academy of Sciences
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Here the authors report that selective optogenetic stimulation of ventral tegmental area (VTA) dopamine neurons in mice produces a powerful arousal response sufficient to restore conscious behaviors, including the righting reflex, during continuous, steady-state general anesthesia. Although previous studies found that VTA dopamine neurons do not appear to play a central role in regulating sleep–wake transitions, their findings demonstrate that selective stimulation of these neurons is sufficient to induce the transition from an unconscious, anesthetized state to an awake state. These results suggest that VTA DA neurons play a critical role in promoting wakefulness.

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To CRISPR and beyond: the evolution of genome editing in stem cells, Regenerative Medicine, Future Medicine

To CRISPR and beyond: the evolution of genome editing in stem cells, Regenerative Medicine, Future Medicine | Genetic Engineering - GEG Tech top picks | Scoop.it
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Here the authors review the history of genome editing in stem cells (including via zinc finger nucleases, transcription activator-like effector nucleases and CRISPR–Cas9), discuss recent developments leading to the implementation of stem cell gene therapies in clinical trials and consider the prospects for future advances in this rapidly evolving field.

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Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array - Nature Biotechnology 

Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array - Nature Biotechnology  | Genetic Engineering - GEG Tech top picks | Scoop.it
Multiplexed genome editing is simplified by harnessing the ability of Cpf1 to process its own pre-crRNA.
BigField GEG Tech's insight:

Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here the scientists show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, they edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.

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Structures and mechanisms of CRISPR RNA-guided effector nucleases

Structures and mechanisms of CRISPR RNA-guided effector nucleases | Genetic Engineering - GEG Tech top picks | Scoop.it
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Over the past few years, intensive studies have provided an atomic view of the crRNA-guided target interference mechanisms in different types of CRISPR-Cas systems. Here, the authors review the recent progress toward structural and mechanistic understanding of the diverse crRNP effector nucleases.

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Regenerative medicine: Engineered iPSCs for cartilage repair - Nature Reviews 

Regenerative medicine: Engineered iPSCs for cartilage repair - Nature Reviews  | Genetic Engineering - GEG Tech top picks | Scoop.it
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Murine induced pluripotent stem cells (iPSCs) lacking IL-1 receptor type 1 (IL-1R1), engineered using the CRISPR/Cas9 system, are able to form cartilaginous tissue that is resistant to IL-1α-mediated inflammation, a new study shows.

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Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors - Nature Medicine

Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors - Nature Medicine | Genetic Engineering - GEG Tech top picks | Scoop.it
BigField GEG Tech's insight:

A genome-wide CRISPR screen reveals that FZD5, but none of the other nine Frizzled receptors encoded in the human genome, is a therapeutic vulnerability of pancreatic and colorectal tumors bearing RNF43 mutations.

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CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes | Genetic Engineering - GEG Tech top picks | Scoop.it
CRISPR-based genome-editing technologies provide powerful tools to study basic biology
and may lead to new treatments for human disease.
BigField GEG Tech's insight:

In this Review, the authors summarize CRISPR-based technologies that enable mammalian genome editing and their various applications. They describe recent developments that extend the generality, DNA specificity, product selectivity, and fundamental capabilities of natural CRISPR systems, and they highlight some of the remarkable advancements in basic research, biotechnology, and therapeutics science that these developments have facilitated.

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miRNAsong: a web-based tool for generation and testing of miRNA sponge constructs in silico

miRNAsong: a web-based tool for generation and testing of miRNA sponge constructs in silico | Genetic Engineering - GEG Tech top picks | Scoop.it

MicroRNA (miRNA) sponges are RNA transcripts containing multiple high-affinity binding sites that associate with and sequester specific miRNAs to prevent them from interacting with their target messenger (m)RNAs.

BigField GEG Tech's insight:

In this study, the scientists introduce microRNA sponge generator and tester (miRNAsong), a freely available web-based tool for generation and in silico testing of miRNA sponges. This tool generates miRNA sponge constructs for specific miRNAs and miRNA families/clusters and tests them for potential binding to miRNAs in selected organisms. Currently, miRNAsong allows for testing of sponge constructs in 219 species covering 35,828 miRNA sequences.

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In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration - Nature

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration - Nature | Genetic Engineering - GEG Tech top picks | Scoop.it
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In this work, the devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, they demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

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Pooled CRISPR screening with single-cell transcriptome read-out

Pooled CRISPR screening with single-cell transcriptome read-out | Genetic Engineering - GEG Tech top picks | Scoop.it
bioRxiv - the preprint server for biology, operated by Cold Spring Harbor Laboratory, a research and educational institution
BigField GEG Tech's insight:

Here the scientists demonstrate CRISPR genome editing together with single-cell RNA sequencing as a new screening paradigm that combines key advantages of pooled and arrayed screens. This approach allowed to link guide-RNA expression to the associated transcriptome responses in thousands of single cells using a straightforward and broadly applicable screening workflow.

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Inhibition of JCPyV infection mediated by targeted viral genome editing using CRISPR/Cas9

Inhibition of JCPyV infection mediated by targeted viral genome editing using CRISPR/Cas9 | Genetic Engineering - GEG Tech top picks | Scoop.it
Progressive multifocal leukoencephalopathy (PML) is a debilitating disease resulting from infection of oligodendrocytes by the JC polyomavirus (JCPyV).
BigField GEG Tech's insight:

In this study, the authors show that the CRISPR/Cas9 system can restrict the JC polyomavirus (JCPyV) life cycle in cultured cells. They utilized CRISPR/Cas9 to target the noncoding control region and the late gene open reading frame of the JCPyV genome. They found significant inhibition of virus replication and viral protein expression in cells recipient of Cas9 together with JCPyV-specific single-guide RNA delivered prior to or after JCPyV infection.

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Using an Inducible CRISPR-dCas9-KRAB Effector System to Dissect Transcriptional Regulation in Human Embryonic Stem Cells

Using an Inducible CRISPR-dCas9-KRAB Effector System to Dissect Transcriptional Regulation in Human Embryonic Stem Cells | Genetic Engineering - GEG Tech top picks | Scoop.it
BigField GEG Tech's insight:

Here, the scientists describe the application of an inducible, RNA-guided, nuclease-deficient Cas9-KRAB system to silence target gene expression in human embryonic stem cells, via KRAB repression at the promoter region. This chapter outlines a detailed protocol for generation of a stable human embryonic stem cell line containing both Sp-dCas9-KRAB and sgRNA, followed by inducible expression of Sp-dCas9-KRAB to analyze functional effects of dCas9-KRAB at target loci in human embryonic stem cells.

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Reactivation of FMR1 by CRISPR/Cas9-Mediated Deletion of the Expanded CGG-Repeat of the Fragile X Chromosome

Reactivation of  FMR1  by CRISPR/Cas9-Mediated Deletion of the Expanded CGG-Repeat of the Fragile X Chromosome | Genetic Engineering - GEG Tech top picks | Scoop.it
BigField GEG Tech's insight:

As a proof-of-principle, the scientists excised the expanded CGG-repeat in both somatic cell hybrids containing the human fragile X chromosome and human FXS iPS cells using the CRISPR/Cas9 genome editing. Their data demonstrate the excision of the expanded CGG-repeat from the fragile X chromosome can result in FMR1 reactivation.

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