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AAV-mediated gene editing lights up the lung.
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Brave new dialogue.
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Bioremediation through microbes: systems biology and metabolic engineering approach.
Today, environmental pollution is a serious problem, and bioremediation can play an important role in cleaning contaminated sites. Remediation strategies, such as chemical and physical approaches, are not enough to mitigate pollution problems because of the continuous generation of novel recalcitrant pollutants due to anthropogenic activities. Bioremediation using microbes is an eco-friendly and socially acceptable alternative to conventional remediation approaches. Many microbes with a bioremediation potential have been isolated and characterized but, in many cases, cannot completely degrade the targeted pollutant or are ineffective in situations with mixed wastes. This review envisages advances in systems biology (SB), which enables the analysis of microbial behavior at a community level under different environmental stresses. By applying a SB approach, crucial preliminary information can be obtained for metabolic engineering (ME) of microbes for their enhanced bioremediation capabilities. This review also highlights the integrated SB and ME tools and techniques for bioremediation purposes.
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Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones.
BACKGROUND: CRISPR/Cas9 editing systems are currently used to generate mutations in a particular gene to mimic a genetic disorder in vitro. Such "disease in a dish" models based on human pluripotent stem cells (hPSCs) offer the opportunity to have access to virtually all cell types of the human body. However, the generation of mutated hPSCs remains fastidious. Current CRISPR/Cas9 editing approaches lead to a mixed cell population containing simultaneously non-edited and a variety of edited cells. These edited hPSCs need therefore to be isolated through manual dilution cloning, which is time-consuming, labor intensive and tedious. METHODS: Following CRISPR/Cas9 edition, we obtained a mixed cell population with various edited cells. We then used a semi-automated robotic platform to isolate single cell-derived clones. RESULTS: We optimized CRISPR/Cas9 editing to knock out a representative gene and developed a semi-automated method for the clonal isolation of edited hPSCs. This method is faster and more reliable than current manual approaches. CONCLUSIONS: This novel method of hPSC clonal isolation will greatly improve and upscale the generation of edited hPSCs required for downstream applications including disease modeling and drug screening.
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Precision BioSciences, Novartis Launch Up-to-$1.5B In Vivo Genome Editing Collaboration.
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Design, Construction, and Validation of Targeted Gene Activation with TREE System in Human Cells.
Genome editing technologies can be diverted into artificial transcription activators. In particular, researchers have improved dCas9-based technologies by tandem-fusing or trans-accumulating effector domains. Previously, we developed a hierarchical effector accumulation system named "TREE," enabling robust activation of target genes even when strongly silenced. In this chapter, we describe our protocol to design, construct, and validate the TREE-mediated target gene activation in cultured human cells.
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A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells.
Ex vivo activation is a prerequisite to reaching adequate levels of gene editing by homology-directed repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here, we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR-Cas9-induced DNA double-strand breaks, enhancing the reconstitution capacity of edited HSPCs. However, this results in lower HDR efficiency, rendering ex vivo culture necessary yet detrimental. Mechanistically, ex vivo activation triggers a multi-step process initiated by p38 mitogen-activated protein kinase (MAPK) phosphorylation, which generates mitogenic reactive oxygen species (ROS), promoting fast cell-cycle progression and subsequent proliferation-induced DNA damage. Thus, p38 inhibition before gene editing delays G1/S transition and expands transcriptionally defined HSCs, ultimately endowing edited cells with superior multi-lineage differentiation, persistence throughout serial transplantation, enhanced polyclonal repertoire, and better-preserved genome integrity. Our data identify proliferative stress as a driver of HSPC dysfunction with fundamental implications for designing more effective and safer gene correction strategies for clinical applications.
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Delivery of genome editors with engineered virus-like particles.
Genome editing technologies have revolutionized biomedical sciences and biotechnology. However, their delivery in vivo remains one of the major obstacles for clinical translation. Here, we introduce various emerging genome editing systems and review different delivery systems have been developed to realize the promise of in vivo gene editing therapies. In particular, we focus on virus-like particles (VLPs), an emerging delivery platform and provide in depth analysis on recent advancements to improve VLPs delivery potential and highlight opportunities for future improvements. To this end, we also provide detail workflows for engineered VLP (eVLP) selection, production, and purification, along with methods for characterization and validation.
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Production of Lentiviral Vectors Encoding the CRISPR-Cas13d System for RNA Targeting.
The CRISPR-Cas13d system has been shown to be a potent tool for target RNA knockdown, offering advantages over DNA-editing systems by providing a non-genotoxic method to modulate gene expression. Efficient delivery of Cas13d and its guide RNAs into target cells is essential to achieve a strong knockdown effect, particularly in cell lines that are difficult to transfect and primary cells. Lentiviral vectors (LVs) are ideal for delivering large gene-editing tools due to their large packaging capacity and ability to transduce both dividing and nondividing cells. Here, we describe a detailed protocol for the production of high-titer, third-generation LVs with a dual-expression cassette for Cas13d and a customizable guide RNA. The method describes the cloning of target-specific guide RNAs, the transfection of HEK293T cells with packaging and transfer plasmids, the harvest and concentration of viral supernatants, and the determination of viral titer. The generated lentiviral particles can be used for stable transduction and efficient RNA knockdown in a broad range of mammalian cell types.
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CRISPR/Cas9 Endonuclease-Mediated Mouse Genome Editing of One-Cell and/or Two-Cell Embryos by Electroporation, and the Use of Rad51 to Enhance Knock-In Allele Homozygosity via Interhomolog Repair Mechanism.
Electroporation of mouse embryos with CRISPR/Cas9 endonuclease tool is a facile and efficient method to edit endogenous genome sequences for generating genetically engineered mouse models (GEMMs). Common genome engineering projects, such as knock-out (KO), conditional knock-out (cKO), point mutation, and small foreign DNA (<1 Kb) knock-in (KI) alleles, can be effectively accomplished with a simple electroporation procedure. The use of electroporation in sequential gene editing at the one-cell (0.7 days post-coitum (dpc)) and at two-cell (1.5 dpc) embryonic stages provides a fast and compelling protocol to safely introduce multiple gene modifications on the same chromosome by limiting chromosomal fractures. In addition, the co-electroporation of the ribonucleoprotein (RNP) complex and single-stranded oligodeoxynucleotide (ssODN) donor DNA with the strand exchange protein Rad51 can significantly increase the number of homozygous founders. Here we describe a comprehensive guideline for mouse embryo electroporation to generate GEMMs and the implementation of Rad51 in RNP/ssODN complex EP medium protocol.
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