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CRISPR/Cas9-Mediated Modification of PTP Expression.
Alteration of protein tyrosine phosphatase (PTP) gene expression is a commonly used approach to experimentally analyze their function in the cell physiology of mammalian cells. Here, exemplified for receptor-type PTPRJ (Dep-1, CD148) and PPTRC (CD45), we provide the CRISPR/Cas9-mediated approaches for their inactivation and transcriptional activation using genome editing. These methods are generally applicable to any other protein of interest.
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Modeling Rare Bone Diseases in Animals.
PURPOSE OF REVIEW: The goal of this review is to highlight some of the considerations involved in creating animal models to study rare bone diseases and then to compare and contrast approaches to creating such models, focusing on the advantages and novel opportunities offered by the CRISPR-Cas system. RECENT FINDINGS: Gene editing after creation of double-stranded breaks in chromosomal DNA is increasingly being used to modify animal genomes. Multiple tools can be used to create such breaks, with the newest ones being based on the bacterial adaptive immune system known as CRISPR/Cas. Advances in gene editing have increased the ease and speed, while reducing the cost, of creating novel animal models of disease. Gene editing has also expanded the number of animal species in which genetic modification can be performed. These changes have significantly increased the options for investigators seeking to model rare bone diseases in animals.
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CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes.
The CRISPR-Cas9 RNA-guided DNA endonuclease has contributed to an explosion of advances in the life sciences that have grown from the ability to edit genomes within living cells. In this Review, we summarize CRISPR-based technologies that enable mammalian genome editing and their various applications. We describe recent developments that extend the generality, DNA specificity, product selectivity, and fundamental capabilities of natural CRISPR systems, and we highlight some of the remarkable advancements in basic research, biotechnology, and therapeutics science that these developments have facilitated.
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Jennifer Doudna.
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Gene editing of SAMHD1 in macrophage-like cells reveals complex relationships between SAMHD1 phospho-regulation, HIV-1 restriction, and cellular dNTP levels.
We introduce BLaER1 cells as an alternative myeloid cell model in combination with CRISPR/Cas9-mediated gene editing to study the influence of sterile α motif and HD domain-containing protein 1 (SAMHD1) T592 phosphorylation on anti-viral restriction and the control of cellular dNTP levels in an endogenous, physiologically relevant context. A proper understanding of the mechanism of the anti-viral function of SAMHD1 will provide attractive strategies aiming at selectively manipulating SAMHD1 without affecting other cellular functions. Even more, our toolkit may inspire further genetic analysis and investigation of restriction factors inhibiting retroviruses and their cellular function and regulation, leading to a deeper understanding of intrinsic anti-viral immunity.
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TriCON: A Carbon-Based Triple-Modal Nanoplatform for Pancreatic Cancer Therapy.
Pancreatic cancer, recognized for its high malignancy and tumor immunosuppressive microenvironment, has been refractory to conventional therapeutic modalities, necessitating the exploration of novel treatment strategies. Among these, reprogramming of the tumor immunosuppressive microenvironment is a promising strategy to enhance the efficacy of tumor immunotherapy. The CRISPR/Cas9 system-based gene editing further offers a viable approach for precise regulation of endogenous gene expression associated with tumor immunosuppression. Current delivery vectors face a trilemma between biosafety profiles, expansion capacity, and targeting accuracy. To this end, we developed a triple-modality therapeutic platform, termed TriCON (Triple Convergent Oncology Nanotherapy), characterized by three core mechanistic attributes: spatiotemporal convergence, stimuli-responsive controllability, and tumor-microenvironment modulatory conductivity. This orchestrated combination of rationally designed gene editing (targeting poliovirus receptor), nano-encapsulated doxorubicin (DOX) chemotherapy, and checkpoint blockade immunotherapy demonstrated enhanced synergistic antitumor activity in pancreatic ductal adenocarcinoma (PDAC) models, achieving tumor regression through enhanced chemotherapy, immunogenic cell death induction, and natural killer (NK) cells activation. The platform achieved superior in vivo gene editing (14.2% PVR editing efficiency) via optimized endosomal escape and CRISPR system release. This triaxial approach establishes a programmable nanotherapeutic paradigm that synergizes gene editing precision with chemo-immunotherapy, offering a novel framework for PDAC treatment.
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Lentivirus pre-packed with Cas9 protein for safer gene editing.
The CRISPR/Cas9 system provides an easy way to edit specific site/s in the genome and thus offers tremendous opportunity for human gene therapy for a wide range of diseases. However, one major concern is off-target effects, particularly with long-term expression of Cas9 nuclease when traditional expression methods such as via plasmid/viral vectors are used. To overcome this limitation, we pre-packaged Cas9 protein (Cas9P LV) in lentiviral particles for transient exposure and showed its effectiveness for gene disruption in cells, including primary T cells expressing specific single guide RNAs (sgRNAs). We then constructed an 'all in one virus' to express sgRNAs in association with pre-packaged Cas9 protein (sgRNA/Cas9P LV). We successfully edited CCR5 in TZM-bl cells by this approach. Using an sgRNA-targeting HIV long terminal repeat, we also were able to disrupt HIV provirus in the J-LAT model of viral latency. Moreover, we also found that pre-packaging Cas9 protein in LV particle reduced off-target editing of chromosome 4:-29134166 locus by CCR5 sgRNA, compared with continued expression from the vector. These results show that sgRNA/Cas9P LV can be used as a safer approach for human gene therapy applications.
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Highly Efficient CRISPR-Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells.
Human PSCs offer tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR-Cas9 technology has expanded this potential by providing a valuable tool for engineering or correcting disease-associated mutations. Because of the high efficiency with which CRISPR-Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele, without indel formation on the non-targeted allele. To overcome this obstacle, we describe the use of two oligonucleotides, one expressing the sequence change, with the other maintaining the normal sequence. In addition, we have streamlined both the transfection and screening methodology to make this protocol efficient with small numbers of cells and to limit the amount of labor-intensive clone passaging. This protocol provides a streamlined and technically simple approach for generating valuable tools to model human disease in stem cells. © 2018 by John Wiley & Sons, Inc.
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Generation of a human induced pluripotent stem cell line with Cas9 driven by Tet-on operator via AAVS1 safe harbor gene-editing.
Human induced pluripotent stem (iPS) cells expressing Cas9 protein are valuable for the pathogenic mechanism study and drug discovery. These cells can be efficiently induced to differentiate into disease cell models with specific mutations through adding designed sgRNAs. Here, we generated a human gene-editable iPS cell line by gene editing method that Cas9 gene driven by Tet-on operator was perfectly integrated into the human AAVS1 safe harbor locus. The established Cas9 expression iPS cell line named as WMUi013-A can express endogenous pluripotent markers, has the ability to differentiate into the three germ layers, and possesses a normal karyotype.
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Geneticists retract study suggesting first CRISPR babies might die early.
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