Inducible crispr

Prostate cancer is a leading diagnosis and major cause of cancer-related deaths in men worldwide. As a typical hormone-responsive disease, prostate cancer is commonly managed with androgen deprivation therapy (ADT) to curb its progression and potential metastasis. Unfortunately, progression to castration-resistant prostate cancer (CRPC), a notably more aggressive phase of the disease, occurs within a timeframe of 2-3 years following ADT. Enzalutamide, a recognized androgen receptor (AR) antagonist, has been employed as a standard of care for men with metastatic castration-resistant prostate cancer (mCRPC) since it was first approved in 2012, due to its ability to prolong survival...

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely applied in animals as an efficient genome editing tool. However, the technique is difficult to implement in fish cell lines partially due to the lack of efficient promoters to drive the expression of both sgRNA and the Cas9 protein within a single vector. In this study, it was indicated that the zebrafish U6 RNA polymerase III (ZFU6) promoter could efficiently induce tyrosinase (tyr) gene editing and lead to loss of retinal pigments when co-injection with Cas9 mRNA in zebrafish embryo...

Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types...

Integration of multiple data sources presents a challenge for accurate prediction of molecular patho-phenotypic features in automated analysis of data from human model systems. Here, we applied a machine learning-based data integration to distinguish patho-phenotypic features at the subcellular level for dilated cardiomyopathy (DCM). We employed a human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model of a DCM mutation in the sarcomere protein troponin T (TnT), TnT-R141W, compared to isogenic healthy (WT) control iPSC-CMs...

In tomato (Solanum lycopersicum), the ripening of fruit is regulated by the selective expression of ripening-related genes, and this procedure is controlled by transcription factors (TFs). In the various plant-specific TF families, the no apical meristem (NAM), Arabidopsis thaliana activating factor 1/2 (ATAF1/2), and cup-shaped cotyledon 2 (CUC2; NAC) TF family stands out and plays a significant function in plant physiological activities, such as fruit ripening (FR). Despite the numerous genes of NAC found in the tomato genome, limited information is available on the effects of NAC members on FR, and there is also a lack of studies on their target genes...

Manganese (Mn) is an essential micronutrient required for various biological processes but excess exposure to Mn can cause neurotoxicity. However, there are few reports regarding the toxicity effect of Mn on the kidney as well as the underlying molecule mechanism. Herein, in vivo experiments were adopted to assess the toxicity effects associated with Mn, and found that chronic Mn treatment induced the injury of glomerular podocytes but not renal tubule in rats. Genome-wide CRISPR/Cas9 knockout screen was then employed to explore the biotargets of the toxic effect of Mn on podocytes...

The CRISPR-Cas9 technology has the potential to revolutionize the treatment of various diseases, including Rett syndrome, by enabling the correction of genes or mutations in human patient cells. However, several challenges need to be addressed before its widespread clinical application. These challenges include the low delivery efficiencies to target cells, the actual efficiency of the genome-editing process, and the precision with which the CRISPR-Cas system operates. Herein, the study presents a Magnetic Nanoparticle-Assisted Genome Editing (MAGE) platform, which significantly improves the transfection efficiency, biocompatibility, and genome-editing accuracy of CRISPR-Cas9 technology...

We analyzed genomic data derived from the prostate cancer of African and European American men in order to identify differences that may contribute to racial disparity of outcome and that could also define novel therapeutic strategies. In addition to analyzing patient derived next generation sequencing data, we performed FISH based confirmatory studies of Chromodomain helicase DNA-binding protein 1 ( CHD1 ) loss on prostate cancer tissue microarrays. We created CRISPR edited, CHD1 deficient prostate cancer cell lines for genomic, drug sensitivity and functional homologous recombination (HR) activity analysis...

Signal transducer and activator of transcription (STAT) 3 is extensively involved in the development, homeostasis and function of immune cells, with STAT3 disruption associated with human immune-related disorders. These roles have been assumed to be due to its canonical mode of action as an inducible transcription factor downstream of multiple cytokines, although alternative non-canonical functional modalities have also been described for STAT3. To further understand the roles of STAT3 gained from lineage-specific mouse knockouts, CRISPR/Cas9 was used to generate mutants of the conserved zebrafish Stat3 protein: a loss of function knockout (KO) mutant and a mutant lacking C-terminal sequences including the transactivation domain (ΔTAD)...

PPM1A is a member of the serine/threonine protein phosphatase family. It can bind to a variety of proteins to dephosphorylate them, and extensively regulates many life activities such as cell growth, cell stress, immune response, and tumor formation. Here we constructed a human induced pluripotent stem cell (hiPSC) line with knockout of PPM1A using CRISPR/Cas9-mediated gene targeting. This cell line exhibits normal karyotype, pluripotency, and trilineage differentiation potential, which could provide a useful cellular resource for exploring the mechanism of PPM1A in regulating downstream signaling pathways and explore the application of PPM1A in anti-tumor and anti-infection...

BACKGROUND: p66Shc, as a redox enzyme, regulates reactive oxygen species (ROS) production in mitochondria and autophagy. However, the mechanisms by which p66Shc affects autophagosome formation are not fully understood. METHODS: p66Shc expression and its location in the trophoblast cells were detected in vivo and in vitro. Small hairpin RNAs or CRISPR/Cas9, RNA sequencing, and confocal laser scanning microscope were used to clarify p66Shc's role in regulating autophagic flux and STING activation...

Pathogenic variants in MYH7 and MYBPC3 account for the majority of hypertrophic cardiomyopathy (HCM). Targeted drugs like myosin ATPase inhibitors have not been evaluated in children. We generate patient and variant-corrected iPSC-cardiomyocytes (CMs) from pediatric HCM patients harboring single variants in MYH7 (V606M; R453C), MYBPC3 (G148R) or digenic variants (MYBPC3 P955fs, TNNI3 A157V). We also generate CMs harboring MYBPC3 mono- and biallelic variants using CRISPR editing of a healthy control. Compared with isogenic and healthy controls, variant-positive CMs show sarcomere disorganization, higher contractility, calcium transients, and ATPase activity...

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) inhibitors have emerged as promising chemotherapeutic drugs primarily against BRCA1/2-associated tumours, known as synthetic lethality. However, recent clinical trials reported patients' survival benefits from PARP inhibitor treatments, irrelevant to homologous recombination deficiency. Therefore, revealing the therapeutic mechanism of PARP inhibitors beyond DNA damage repair is urgently needed, which can facilitate precision medicine. METHODS: A CRISPR-based knock-in technology was used to establish stable BRCA1 mutant cancer cells...

During neuroinflammation, the proinflammatory cytokine interleukin-1β (IL-1β) impacts blood-brain barrier (BBB) function by disrupting brain endothelial tight junctions, promoting vascular permeability, and increasing transmigration of immune cells. Here, we examined the effects of Il-1β on the in vivo initiation of BBB development. We generated doxycycline-inducible transgenic zebrafish to secrete Il-1β in the CNS. To validate the utility of our model, we showed Il-1β dose-dependent mortality, recruitment of neutrophils, and expansion of microglia...

INTRODUCTION: Protocadherin-19 ( PCDH19 )-Clustering Epilepsy (PCE) is a developmental and epileptic encephalopathy caused by loss-of-function variants of the PCDH19 gene on the X-chromosome. PCE affects females and mosaic males while male carriers are largely spared. Mosaic expression of the cell adhesion molecule PCDH19 due to random X-chromosome inactivation is thought to impair cell-cell interactions between mutant and wild type PCDH19 -expressing cells to produce the disease. Progress has been made in understanding PCE using rodent models or patient induced pluripotent stem cells (iPSCs)...

The rational development of small-molecule degraders (e.g., proteolysis targeting chimeras) remains a challenge as the rate-limiting steps that determine degrader efficiency are largely unknown. Standard methods in the field of targeted protein degradation mostly rely on classical, low-throughput endpoint assays such as western blots or quantitative proteomics. Here we applied NanoLuciferase- and HaloTag-based screening technologies to determine the kinetics and stability of small-molecule-induced ternary complex formation between a protein of interest and a selected E3 ligase...

Under hypoxia, hypoxia-inducible factor (HIF)-1 regulates hypoxia-inducible genes, such as vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2. It is an oxygen-dependent transcriptional activator that plays a crucial role in tumor angiogenesis and mammalian embryo development. It is a heterodimeric protein comprising a constitutively expressed HIF-1β subunit and the highly regulated HIF-1α subunits. Using CRISPR-Cas9 genome editing, we generated biallelic HIF-1α mutants in human induced pluripotent stem cells (hiPSCs)...

Oncogenic KRAS impairs anti-tumor immune responses. As effective strategies to combine KRAS inhibitors and immunotherapies have so far proven elusive, a better understanding of how oncogenic KRAS drives immune evasion is needed to identify approaches that could sensitize KRAS-mutant lung cancer to immunotherapy. In vivo CRISPR-Cas9 screening in an immunogenic murine lung cancer model identified mechanisms by which oncogenic KRAS promotes immune evasion, most notably via upregulation of immunosuppressive cyclooxygenase-2 (COX-2) in cancer cells...

Anaphylaxis is a severe life-threatening hypersensitivity reaction induced by mast cell degranulation. Among the various mediators of mast cells, little is known about the role of tryptase. Therefore, we aimed to elucidate the role of protease-activating receptor-2 (PAR-2), a receptor activated by tryptase, in murine anaphylactic models using PAR-2-deficient mice and newly generated tryptase-deficient mice. Anaphylaxis was induced by IgE-dependent and IgE-independent mast cell degranulation in mice. PAR-2 deficiency exacerbated the decrease in body temperature and hypotension during anaphylaxis; however, the number of skin mast cells, degree of mast cell degranulation, and systemic and local vascular hyperpermeability were comparable in PAR-2 knockout and wild-type mice...

Integration of a point mutation to correct or edit a gene requires the repair of the CRISPR-Cas9-induced double-strand break by homology-directed repair (HDR). This repair pathway is more active in late S and G2 phases of the cell cycle, whereas the competing pathway of nonhomologous end-joining (NHEJ) operates throughout the cell cycle. Accordingly, modulation of the cell cycle by chemical perturbation or simply by the timing of gene editing to shift the editing toward the S/G2 phase has been shown to increase HDR rates...