Generation of Dopamine Transporter (DAT)-mCherry Knock-in Rats by CRISPR-Cas9 Genome Editing.

Midbrain dopaminergic neurons respond to rewards and have a crucial role in positive motivation and pleasure. Electrical stimulation of dopaminergic neurons and/or their axonal fibers and arborization has been often used to motivate animals to perform cognitive tasks. Still, the electrical stimulation is incompatible with electrophysiological recordings. In this light, optical stimulation following artificial expression of channelrhodopsin-2 (ChR2) in the cell membrane has been also used, but the expression level of ChR2 varies among researchers. Thus, we attempted to stably express ChR2 fused with a red fluorescence protein, mCherry, in dopaminergic neurons. Since dopamine transporter (DAT) gene is known as a marker for dopaminergic neurons, we inserted ChR2-mCherry into the downstream of the DAT gene locus of the rat genome by clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) genome editing and created DAT-ChR2-mCherry knock-in rats. Immunohistochemistry showed that ChR2-mCherry was expressed in dopaminergic neurons in homozygote knock-in rats, whereas whole-cell recordings revealed that ChR2-mCherry-positive neurons did not fire action potentials upon blue light stimulation, indicating that ChR2 was not functional for optogenetics. Nevertheless, fluorescent labeling of dopaminergic neurons mediated by mCherry could help characterize them physiologically and histologically.

CRISPR-Cas9-Edited SNCA Knockout Human Induced Pluripotent Stem Cell-Derived Dopaminergic Neurons and Their Vulnerability to Neurotoxicity.

Parkinson's disease (PD) is an age-related disorder with selective dopaminergic (DA) neuronal degeneration in the substantia nigra pars compacta. The presence of mainly α-synuclein-composed Lewy bodies in DA neurons is among the disease hallmarks in the brain of patients with PD. Human induced pluripotent stem cells (hiPSCs) are powerful tools to investigate PD pathophysiology and understand its molecular and cellular mechanisms better. In this study, we generated an α-synuclein-null hiPSC line introducing a nonsense mutation in the α-synuclein-encoding SNCA alleles using clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9)-mediated gene editing. Our Western blotting analysis revealed the lack of α-synuclein protein expression in SNCA knockout hiPSC-derived cells. In addition, SNCA knockout hiPSCs retained healthy cell morphology, undifferentiated marker gene (e.g., NANOG, POU5F1, and SOX2) expression, and differentiation ability (based on the marker gene expression levels of the three germ layers). Finally, SNCA knockout hiPSC-derived DA neurons exhibited reduced vulnerability to the DA neurotoxin, 1-methyl-4-phenylpyridinium. In conclusion, the SNCA knockout hiPSC line we generated would provide a useful experimental tool for studying the physiological and pathological role of α-synuclein in PD.

A New Era of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Protein 9 Gene Editing Technology in Cardiovascular Diseases: Opportunities, Challenges, and Perspectives.

Cardiovascular diseases (CVDs) remain major causes of global mortality in the world. Genetic approaches have succeeded in the discovery of the molecular basis of an increasing number of cardiac diseases. Genome-editing strategies are one of the most effective methods for assisting therapeutic approaches. Potential therapeutic methods of correcting disease-causing mutations or of knocking out specific genes as approaches for the prevention of CVDs have gained substantial attention using genome-editing techniques. Recently, the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has become the most widely used genome-editing technology in molecular biology due to its benefits such as simple design, high efficiency, good repeatability, short cycle, and cost-effectiveness. In the present review, we discuss the possibilities of applying the CRISPR/Cas9 genome-editing tool in the CVDs.

Genome-Wide Knockout Screen Identifies EGLN3 Involving in Ammonia Neurotoxicity.

Hepatic encephalopathy (HE) is a brain dysfunction associated with poor quality of life, increased morbidity and mortality. The pathogenesis of HE is still not fully clarified and effective therapeutic strategies are imperative. Among multiple factors that contribute to the pathophysiological process of HE, ammonia neurotoxicity is thought to be central in the pathogenesis of HE. Therefore, in this study, we subjected SH-SY5Y cells to ammonia insult and performed a pooled genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) knockout screen to unveil the underlying molecular mechanisms of ammonia neurotoxicity and discover new potential therapeutic targets for HE. We found that EGLN3 (egl-9 family hypoxia-inducible factor 3) UCP3,GTPBP5, OR4D11 and SDR9C7 with 6 unique sgRNAs may contribute to protection against ammonia injury, while EGLN3 may be most related to ammonia resistance. We knocked down EGLN3 by transfecting neurons with specific shRNA lentivirus and confirmed that EGLN3 knockdown decreased ammonia-induced caspase-3 activation and apoptosis. We also demonstrated that EGLN3 knockdown ameliorated ammonia induced decreased expression of Bcl-2, increased expression of Bax and inhibited release of cytochrome c into the cytosol in neurons, suggesting that EGLN3 inhibition protected against ammonia induced apoptosis through mitochondrial dependent apoptosis pathway. Future therapeutic strategies regulating EGLN3 may be applied to the management of HE.

CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX.

Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies.

Can genetic engineering-based methods for gene function identification be eclipsed by genome editing in plants? A comparison of methodologies.

Finding and explaining the functions of genes in plants have promising applications in crop improvement and bioprospecting and hence, it is important to compare various techniques available for gene function identification in plants. Today, the most popular technology among researchers to identify the functions of genes is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-based genome editing method. But by no means can we say that CRISPR/Cas9 is the go-to method for all purposes. It comes with its own baggage. Researchers will agree and have lived through at least seven more technologies deployed to find the functions of genes, which come under three umbrellas: 1. genetic engineering, 2. transient expression, and 3. chemical/physical mutagenesis. Each of the methods evolved when the previous one ran into an insurmountable problem. In this review, we compare the eight technologies against one another on 14 parameters. This review lays bare the pros and cons, and similarities and dissimilarities of various methods. Every method comes with its advantages and disadvantages. For example, the CRISPR/Cas9-based genome editing is an excellent method for modifying gene sequences, creating allelic versions of genes, thereby aiding the understanding of gene function. But it comes with the baggage of unwanted or off-target mutations. Then, we have methods based on random or targeted knockout of the gene, knockdown, and overexpression of the gene. Targeted disruption of genes is required for complete knockout of gene function, which may not be accomplished by editing. We have also discussed the strategies to overcome the shortcomings of the targeted gene-knockout and the CRISPR/Cas9-based methods. This review serves as a comprehensive guide towards the understanding and comparison of various technologies available for gene function identification in plants and hence, it will find application for crop improvement and bioprospecting related research.

Genome-Wide Analysis of Off-Target CRISPR/Cas9 Activity in Single-Cell-Derived Human Hematopoietic Stem and Progenitor Cell Clones.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated genome editing holds remarkable promise for the treatment of human genetic diseases. However, the possibility of off-target Cas9 activity remains a concern. To address this issue using clinically relevant target cells, we electroporated Cas9 ribonucleoprotein (RNP) complexes (independently targeted to two different genomic loci, the CXCR4 locus on chromosome 2 and the AAVS1 locus on chromosome 19) into human mobilized peripheral blood-derived hematopoietic stem and progenitor cells (HSPCs) and assessed the acquisition of somatic mutations in an unbiased, genome-wide manner via whole genome sequencing (WGS) of single-cell-derived HSPC clones. Bioinformatic analysis identified >20,000 total somatic variants (indels, single nucleotide variants, and structural variants) distributed among Cas9-treated and non-Cas9-treated control HSPC clones. Statistical analysis revealed no significant difference in the number of novel non-targeted indels among the samples. Moreover, data analysis showed no evidence of Cas9-mediated indel formation at 623 predicted off-target sites. The median number of novel single nucleotide variants was slightly elevated in Cas9 RNP-recipient sample groups compared to baseline, but did not reach statistical significance. Structural variants were rare and demonstrated no clear causal connection to Cas9-mediated gene editing procedures. We find that the collective somatic mutational burden observed within Cas9 RNP-edited human HSPC clones is indistinguishable from naturally occurring levels of background genetic heterogeneity.

Depletion of essential isoprenoids and ER stress induction following acute liver-specific deletion of HMG-CoA reductase.

HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing nonsterol isoprenoids, such as dolichol, ubiquinone, and farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on nonsterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-Cre) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of Hmgcr by AAV-Cre resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the nonsterol isoprenoids, dolichol and ubiquinone. At the cellular level, Hmgcr-null hepatocytes showed ER stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress. Using AAV-CRISPR, we somatically disrupted dehydrodolichyl diphosphate synthase subunit (Dhdds), encoding a branch point enzyme required for dolichol biosynthesis. Dhdds-null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of Hmgcr and Dhdds synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.

CRISPR/Cas9-mediated whole genomic wide knockout screening identifies mitochondrial ribosomal proteins involving in oxygen-glucose deprivation/reperfusion resistance.

Recanalization therapy by intravenous thrombolysis or endovascular therapy is critical for the treatment of cerebral infarction. However, the recanalization treatment will also exacerbate acute brain injury and even severely threatens human life due to the reperfusion injury. So far, the underlying mechanisms for cerebral ischaemia-reperfusion injury are poorly understood and effective therapeutic interventions are yet to be discovered. Therefore, in the research, we subjected SK-N-BE(2) cells to oxygen-glucose deprivation/reperfusion (OGDR) insult and performed a pooled genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) knockout screen to discover new potential therapeutic targets for cerebral ischaemia-reperfusion injury. We used Metascape to identify candidate genes which might involve in OGDR resistance. We found that the genes contributed to OGDR resistance were primarily involved in neutrophil degranulation, mitochondrial translation, and regulation of cysteine-type endopeptidase activity involved in apoptotic process and response to oxidative stress. We then knocked down some of the identified candidate genes individually. We demonstrated that MRPL19, MRPL32, MRPL52 and MRPL51 inhibition increased cell viability and attenuated OGDR-induced apoptosis. We also demonstrated that OGDR down-regulated the expression of MRPL19 and MRPL51 protein. Taken together, our data suggest that genome-scale screening with Cas9 is a reliable tool to analyse the cellular systems that respond to OGDR injury. MRPL19 and MRPL51 contribute to OGDR resistance and are supposed to be promising targets for the treatment of cerebral ischaemia-reperfusion damage.

Argonaute proteins: Structural features, functions and emerging roles.

Argonaute proteins are highly conserved in almost all organisms. They not only involve in the biogenesis of small regulatory RNAs, but also regulate gene expression and defend against foreign pathogen invasion via small RNA-mediated gene silencing pathways. As a key player in these pathways, the abnormal expression and/or mis-modifications of Argonaute proteins lead to the disorder of small RNA biogenesis and functions, thus influencing multiply biological processes and disease development, especially cancer. In this review, we focus on the post-translational modifications and novel functions of Argonaute proteins in alternative splicing, host defense and genome editing.

Glial cell line-derived neurotrophic factor receptor Rearranged during transfection agonist supports dopamine neurons in Vitro and enhances dopamine release In Vivo.

BACKGROUND: Motor symptoms of Parkinson's disease (PD) are caused by degeneration and progressive loss of nigrostriatal dopamine neurons. Currently, no cure for this disease is available. Existing drugs alleviate PD symptoms but fail to halt neurodegeneration. Glial cell line-derived neurotrophic factor (GDNF) is able to protect and repair dopamine neurons in vitro and in animal models of PD, but the clinical use of GDNF is complicated by its pharmacokinetic properties. The present study aimed to evaluate the neuronal effects of a blood-brain-barrier penetrating small molecule GDNF receptor Rearranged in Transfection agonist, BT13, in the dopamine system. METHODS: We characterized the ability of BT13 to activate RET in immortalized cells, to support the survival of cultured dopamine neurons, to protect cultured dopamine neurons against neurotoxin-induced cell death, to activate intracellular signaling pathways both in vitro and in vivo, and to regulate dopamine release in the mouse striatum as well as BT13's distribution in the brain. RESULTS: BT13 potently activates RET and downstream signaling cascades such as Extracellular Signal Regulated Kinase and AKT in immortalized cells. It supports the survival of cultured dopamine neurons from wild-type but not from RET-knockout mice. BT13 protects cultured dopamine neurons from 6-Hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+ )-induced cell death only if they express RET. In addition, BT13 is absorbed in the brain, activates intracellular signaling cascades in dopamine neurons both in vitro and in vivo, and also stimulates the release of dopamine in the mouse striatum. CONCLUSION: The GDNF receptor RET agonist BT13 demonstrates the potential for further development of novel disease-modifying treatments against PD. © 2019 International Parkinson and Movement Disorder Society.

Epithelial Smad4 Deletion Up-Regulates Inflammation and Promotes Inflammation-Associated Cancer.

Background & Aims: Chronic inflammation is a predisposing condition for colorectal cancer. Many studies to date have focused on proinflammatory signaling pathways in the colon. Understanding the mechanisms that suppress inflammation, particularly in epithelial cells, is critical for developing therapeutic interventions. Here, we explored the roles of transforming growth factor ß (TGFß) family signaling through SMAD4 in colonic epithelial cells. Methods: The Smad4 gene was deleted specifically in adult murine intestinal epithelium. Colitis was induced by 3 rounds of dextran sodium sulfate in drinking water, after which mice were observed for up to 3 months. Nontransformed mouse colonocyte cell lines and colonoid cultures and human colorectal cancer cell lines were analyzed for responses to TGFß1 and bone morphogenetic protein 2. Results: Dextran sodium sulfate treatment was sufficient to drive carcinogenesis in mice lacking colonic Smad4 expression, with resulting tumors bearing striking resemblance to human colitis-associated carcinoma. Loss of SMAD4 protein was observed in 48% of human colitis-associated carcinoma samples as compared with 19% of sporadic colorectal carcinomas. Loss of Smad4 increased the expression of inflammatory mediators within nontransformed mouse colon epithelial cells in vivo. In vitro analysis of mouse and human colonic epithelial cell lines and organoids indicated that much of this regulation was cell autonomous. Furthermore, TGFß signaling inhibited the epithelial inflammatory response to proinflammatory cytokines. Conclusions: TGFß suppresses the expression of proinflammatory genes in the colon epithelium, and loss of its downstream mediator, SMAD4, is sufficient to initiate inflammation-driven colon cancer. Transcript profiling: GSE100082.

Steroidogenic factor-1 hypermethylation in maternal rat blood could serve as a biomarker for intrauterine growth retardation.

Intrauterine growth retardation (IUGR) is a common obstetric complication lacking an optimal method for prenatal screening. DNA methylation profile in maternal blood holds significant promise for prenatal screening. Here, we aimed to screen out potential IUGR biomarkers in maternal blood from the perspective of DNA methylation. The IUGR rat model was established by prenatal maternal undernutrition. High-throughput bisulfite sequencing of genomic DNA methylation followed by functional clustering analysis for differentially methylated region (DMR)-associated genes demonstrated that genes regulating transcription had the most significantly changed DNA methylation status in maternal blood with IUGR. Genes about apoptosis and placental development were also changed. Besides increased placental apoptosis, IUGR rats demonstrated the same hypermethylated CpG sites of steroidogenic factor-1 (SF-1, a DMR-associated transcription factor about placenta) promoter in maternal blood and placentae. Further, ff1b, the SF-1 ortholog, was knocked out in zebrafish by CRISPR/Cas9 technology. The knock-out zebrafish demonstrated developmental inhibition and increased IUGR rates, which confirmed the role of SF-1 in IUGR development. Finally, hypermethylated SF-1 was observed in human maternal blood of IUGR. This study firstly presented distinct DNA methylation profile in maternal blood of IUGR and showed hypermethylated SF-1 could be a potential IUGR biomarker in maternal rat blood.

CRISPR-Cas9 technology and its application in haematological disorders.

The recent advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system for precise genome editing has revolutionized methodologies in haematology and oncology studies. CRISPR-Cas9 technology can be used to remove and correct genes or mutations, and to introduce site-specific therapeutic genes in human cells. Inherited haematological disorders represent ideal targets for CRISPR-Cas9-mediated gene therapy. Correcting disease-causing mutations could alleviate disease-related symptoms in the near future. The CRISPR-Cas9 system is also a useful tool for delineating molecular mechanisms involving haematological malignancies. Prior to the use of CRISPR-Cas9-mediated gene correction in humans, appropriate delivery systems with higher efficiency and specificity must be identified, and ethical guidelines for applying the technology with controllable safety must be established. Here, the latest applications of CRISPR-Cas9 technology in haematological disorders, current challenges and future directions are reviewed and discussed.