scholarly journals CRISPR-Cas Systems: Prospects for Use in Medicine

2020 ◽  
Vol 10 (24) ◽  
pp. 9001
Author(s):  
Marina V. Zaychikova ◽  
Valery N. Danilenko ◽  
Dmitry A. Maslov
Keyword(s):  
Gene Expression ◽  
Gene Expression Regulation ◽  
Genetic Diseases ◽  
Nuclease Activity ◽  
Model Systems ◽  
Inherited Disorders ◽  
Exogenous Dna ◽  
Dna Plasmid ◽  
Genomic Editing ◽  
Direct Use

CRISPR-Cas systems, widespread in bacteria and archaea, are mainly responsible for adaptive cellular immunity against exogenous DNA (plasmid and phage). However, the latest research shows their involvement in other functions, such as gene expression regulation, DNA repair and virulence. In recent years, they have undergone intensive research as convenient tools for genomic editing, with Cas9 being the most commonly used nuclease. Gene editing may be of interest in biotechnology, medicine (treatment of inherited disorders, cancer, etc.), and in the development of model systems for various genetic diseases. The dCas9 system, based on a modified Cas9 devoid of nuclease activity, called CRISPRi, is widely used to control gene expression in bacteria for new drug biotargets validation and is also promising for therapy of genetic diseases. In addition to direct use for genomic editing in medicine, CRISPR-Cas can also be used in diagnostics, for microorganisms’ genotyping, controlling the spread of drug resistance, or even directly as “smart” antibiotics. This review focuses on the main applications of CRISPR-Cas in medicine, and challenges and perspectives of these approaches.

scholarly journals A Cre-dependent CRISPR/dCas9 activation system for gene expression regulation in neurons

2020 ◽  
Author(s):  
Nancy V. N. Carullo ◽  
Jasmin S. Revanna ◽  
Jennifer J. Tuscher ◽  
Allison J. Bauman ◽  
Jeremy J. Day
Keyword(s):  
Gene Expression ◽  
Viral Vectors ◽  
Gene Expression Regulation ◽  
Gene Induction ◽  
Model Systems ◽  
Model Organisms ◽  
Reading Frame ◽  
Specific Targeting ◽  
Technological Advances ◽  
Inducible Genes

Site-specific genetic and epigenetic targeting of distinct cell populations is a central goal in molecular neuroscience and is crucial to understand the gene regulatory mechanisms that underlie complex phenotypes and behaviors. While recent technological advances have enabled unprecedented control over gene expression, many of these approaches are focused on selected model organisms and/or require labor-intensive customizations for different applications. The simplicity and modularity of CRISPR-based systems have transformed this aspect of genome editing, providing a variety of possible applications and targets. However, there are currently few available tools for cell-selective CRISPR regulation in neurons. Here, we optimized a CRISPR activation (CRISPRa) system for Cre recombinase-dependent, cell type-specific targeting. Unexpectedly, CRISPR systems based on a traditional double-floxed inverted open reading frame (DIO) strategy exhibited leaky target gene induction in the absence of Cre. Therefore, we developed an intron-containing Cre-dependent CRISPRa system (SVI-DIO-dCas9-VPR) that alleviated leaky gene induction and outperformed the traditional DIO system at endogenous genes in both HEK293T cells and rat primary neuron cultures. Using gene-specific CRISPR sgRNAs, we demonstrate that SVI-DIO-dCas9-VPR can activate highly inducible genes (GRM2 and Tent5b) as well as moderately inducible genes (Sstr2 and Gadd45b) in a Cre-specific manner. These results provide a robust framework for Cre-dependent CRISPR-dCas9 approaches across different model systems, and will enable cell-specific targeting when combined with common Cre driver lines or Cre delivery via viral vectors.

scholarly journals Genotype-free individual genome reconstruction of Multiparental Population Models by RNA sequencing data

2020 ◽  
Author(s):  
Kwangbom Choi ◽  
Hao He ◽  
Daniel M. Gatti ◽  
Vivek M. Philip ◽  
Narayanan Raghupathy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Gene Expression Regulation ◽  
Agricultural Research ◽  
Model Systems ◽  
Specific Gene ◽  
Rna Seq ◽  
Sequencing Data ◽  
Individual Genome ◽  
Genome Reconstruction

AbstractMulti-parent populations (MPPs), genetically segregating model systems derived from two or more inbred founder strains, are widely used in biomedical and agricultural research. Gene expression profiling by direct RNA sequencing (RNA-Seq) is commonly applied to MPPs to investigate gene expression regulation and to identify candidate genes. In genetically diverse populations, including most MPPs, quantification of gene expression is improved when the RNA-Seq reads are aligned to individualized transcriptomes that incorporate known polymorphic loci. However, the process of constructing and analyzing individual genomes can be computationally demanding and error prone. We propose a new approach, genome reconstruction by RNA-Seq (GBRS), that relies on simultaneous alignment of RNA-Seq reads to the founder strain transcriptomes. GBRS can reconstruct the diploid genome of each individual and quantify both total and allele-specific gene expression. We demonstrate that GBRS performs as well as methods that rely on high-density genotyping arrays to reconstruct the founder haplotype mosaic of MPP individuals. Using GBRS in addition to other genotyping methods provides quality control for detecting sample mix-ups and improves power to detect expression quantitative trait loci. GBRS software is freely available at https://github.com/churchill-lab/gbrs.

scholarly journals CFTR Cooperative Cis-Regulatory Elements in Intestinal Cells

2021 ◽  
Vol 22 (5) ◽  
pp. 2599
Author(s):  
Mégane Collobert ◽  
Ozvan Bocher ◽  
Anaïs Le Nabec ◽  
Emmanuelle Génin ◽  
Claude Férec ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cystic Fibrosis ◽  
Gene Regulation ◽  
Genetic Diseases ◽  
Regulatory Elements ◽  
Cftr Gene ◽  
Intestinal Cells ◽  
Cooperative Effects ◽  
Cis Acting ◽  
Study Techniques

About 8% of the human genome is covered with candidate cis-regulatory elements (cCREs). Disruptions of CREs, described as “cis-ruptions” have been identified as being involved in various genetic diseases. Thanks to the development of chromatin conformation study techniques, several long-range cystic fibrosis transmembrane conductance regulator (CFTR) regulatory elements were identified, but the regulatory mechanisms of the CFTR gene have yet to be fully elucidated. The aim of this work is to improve our knowledge of the CFTR gene regulation, and to identity factors that could impact the CFTR gene expression, and potentially account for the variability of the clinical presentation of cystic fibrosis as well as CFTR-related disorders. Here, we apply the robust GWAS3D score to determine which of the CFTR introns could be involved in gene regulation. This approach highlights four particular CFTR introns of interest. Using reporter gene constructs in intestinal cells, we show that two new introns display strong cooperative effects in intestinal cells. Chromatin immunoprecipitation analyses further demonstrate fixation of transcription factors network. These results provide new insights into our understanding of the CFTR gene regulation and allow us to suggest a 3D CFTR locus structure in intestinal cells. A better understand of regulation mechanisms of the CFTR gene could elucidate cases of patients where the phenotype is not yet explained by the genotype. This would thus help in better diagnosis and therefore better management. These cis-acting regions may be a therapeutic challenge that could lead to the development of specific molecules capable of modulating gene expression in the future.

scholarly journals Sex-dependent dynamics of metabolism in primary mouse hepatocytes

2021 ◽  
Author(s):  
Luise Hochmuth ◽  
Christiane Körner ◽  
Fritzi Ott ◽  
Daniela Volke ◽  
Kaja Blagotinšek Cokan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Sexual Dimorphism ◽  
New Drugs ◽  
Model Systems ◽  
Specific Gene ◽  
Primary Hepatocytes ◽  
Alcoholic Fatty Liver ◽  
Male And Female ◽  
Primary Mouse

AbstractThe liver is one of the most sexually dimorphic organs. The hepatic metabolic pathways that are subject to sexual dimorphism include xenobiotic, amino acid and lipid metabolism. Non-alcoholic fatty liver disease and hepatocellular carcinoma are among diseases with sex-dependent prevalence, progression and outcome. Although male and female livers differ in their abilities to metabolize foreign compounds, including drugs, sex-dependent treatment and pharmacological dynamics are rarely applied in all relevant cases. Therefore, it is important to consider hepatic sexual dimorphism when developing new treatment strategies and to understand the underlying mechanisms in model systems. We isolated primary hepatocytes from male and female C57BL6/N mice and examined the sex-dependent transcriptome, proteome and extracellular metabolome parameters in the course of culturing them for 96 h. The sex-specific gene expression of the general xenobiotic pathway altered and the female-specific expression of Cyp2b13 and Cyp2b9 was significantly reduced during culture. Sex-dependent differences of several signaling pathways increased, including genes related to serotonin and melatonin degradation. Furthermore, the ratios of male and female gene expression were inversed for other pathways, such as amino acid degradation, beta-oxidation, androgen signaling and hepatic steatosis. Because the primary hepatocytes were cultivated without the influence of known regulators of sexual dimorphism, these results suggest currently unknown modulatory mechanisms of sexual dimorphism in vitro. The large sex-dependent differences in the regulation and dynamics of drug metabolism observed during cultivation can have an immense influence on the evaluation of pharmacodynamic processes when conducting initial preclinical trials to investigate potential new drugs.

scholarly journals A gain-of-function single nucleotide variant creates a new promoter which acts as an orientation-dependent enhancer-blocker

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yavor K. Bozhilov ◽  
Damien J. Downes ◽  
Jelena Telenius ◽  
A. Marieke Oudelaar ◽  
Emmanuel N. Olivier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genetic Diseases ◽  
Regulatory Elements ◽  
Base Change ◽  
Dependent Manner ◽  
Globin Genes ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Super Enhancer ◽  
Single Base Change

AbstractMany single nucleotide variants (SNVs) associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. Here we show that a single base change in an otherwise unremarkable region of the human α-globin cluster creates an entirely new promoter and an associated unidirectional transcript. This SNV downregulates α-globin expression causing α-thalassaemia. Of note, the new promoter lying between the α-globin genes and their associated super-enhancer disrupts their interaction in an orientation-dependent manner. Together these observations show how both the order and orientation of the fundamental elements of the genome determine patterns of gene expression and support the concept that active genes may act to disrupt enhancer-promoter interactions in mammals as in Drosophila. Finally, these findings should prompt others to fully evaluate SNVs lying outside of known regulatory elements as causing changes in gene expression by creating new regulatory elements.

Gerea: Prediction Of Gene Expression Regulators From Transcriptome Profiling Data To Transition Networks

2021 ◽  
Vol 16 ◽  
Author(s):  
Min Yao ◽  
Caiyun Jiang ◽  
Chenglong Li ◽  
Yongxia Li ◽  
Shan Jiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Networks ◽  
Target Gene ◽  
Gene Expression Regulation ◽  
Transcriptome Profiling ◽  
Expression Regulation ◽  
Mouse Gene ◽  
Mouse Gene Expression ◽  
Causality Inference ◽  
Identify Gene Expression

Background: Mammalian genes are regulated at the transcriptional and post-transcriptional levels. These mechanisms may involve the direct promotion or inhibition of transcription via a regulator or post-transcriptional regulation through factors such as micro (mi)RNAs. Objective: This study aimed to construct gene regulation relationships modulated by causality inference-based miRNA-(transition factor)-(target gene) networks and analyze gene expression data to identify gene expression regulators. Methods: Mouse gene expression regulation relationships were manually curated from literature using a text mining method which was then employed to generate miRNA-(transition factor)-(target gene) networks. An algorithm was then introduced to identify gene expression regulators from transcriptome profiling data by applying enrichment analysis to these networks. Results: A total of 22,271 mouse gene expression regulation relationships were curated for 4,018 genes and 242 miRNAs. GEREA software was developed to perform the integrated analyses. We applied the algorithm to transcriptome data for synthetic miR-155 oligo-treated mouse CD4+ T-cells and confirmed that miR-155 is an important network regulator. The software was also tested on publicly available transcriptional profiling data for Salmonella infection, resulting in the identification of miR-125b as an important regulator. Conclusion: The causality inference-based miRNA-(transition factor)-(target gene) networks serve as a novel resource for gene expression regulation research, and GEREA is an effective and useful adjunct to the currently available methods. The regulatory networks and the algorithm implemented in the GEREA software package are available under a free academic license at website : http://www.thua45.cn/gerea.

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