Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

10.3791/61618 ◽  
2021 ◽  
Author(s):  
Sean Graham ◽  
Lei Gao ◽  
Rui Wang
Keyword(s):  
Gene Function ◽  
Target Gene ◽  
Agonistic Antibody ◽  
Colitis Model

scholarly journals Poor concordance of floxed sequence recombination in single neural stem cells: Implications for cell autonomous studies

2019 ◽  
Author(s):  
Tyler Joseph Dause ◽  
Elizabeth Diana Kirby
Keyword(s):  
Gene Function ◽  
Target Gene ◽  
Single Cells ◽  
Gene Recombination ◽  
Cell Level ◽  
Fluorescent Reporter ◽  
Site Specific ◽  
Poor Predictor ◽  
Adult Cell ◽  
Stem And Progenitor Cells

SummaryTo manipulate target gene function in specific adult cell populations, tamoxifen-dependent CreERT2 is widely used to drive inducible, site-specific recombination of LoxP flanked sequences. In studies of cell autonomous target gene function, it is common practice to combine these CreERT2-lox systems with a ubiquitously-expressed stop-floxed fluorescent reporter gene to identify single cells supposedly undergoing target gene recombination. Here, we studied the reliability of using Cre-induced recombination of one gene to predict recombination in another gene at the single cell level in adult hippocampal neural stem and progenitor cells. Using two separate stop-floxed reporters plus a Nestin promoter-driven CreERT2, we found that, in individual cells, expression of one reporter was a poor predictor of expression of the other. These findings imply that use of stop-floxed reporters to investigate cell autonomous gene function is likely to lead to false conclusions because recombination in separate genes shows poor concordance in individual cells.

scholarly journals DNA Methylation of miR-122 Aggravates Oxidative Stress in Colitis Targeting SELENBP1 Partially by p65NF-κB Signaling

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Jianan Bai ◽  
Junchi Yu ◽  
Jintian Wang ◽  
Bingyan Xue ◽  
Na He ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Methylation ◽  
Target Gene ◽  
Bisulfite Sequencing ◽  
Cpg Islands ◽  
Colonic Tissue ◽  
Tissue Samples ◽  
Benzene Sulfonic Acid ◽  
Ht 29 ◽  
Colitis Model

Aberrant microRNA (miRNA) expressions contribute to the development and progression of various diseases, including Crohn’s disease (CD). However, the accurate mechanisms of miRNAs in CD are definitely unclear. We employed colonic tissue samples from normal volunteers and CD patients, an acute mice colitis model induced by 2,4,6-trinitro-benzene-sulfonic acid (TNBS), and a cellular oxidative stress model induced by H2O2 in HT-29 cells to determine the effects of oxidative stress on expressions of miR-122, selenium-binding protein 1 (SELENBP1, SBP1), p65 nuclear factor κB (p65NF-κB) signaling, and DNA methylation. We found that SBP1 was mainly located on epithelial cells and was significantly increased in patients with active CD. SBP1 was the target gene of miR-122. miR-122 expression was downregulated while SBP1 expression was upregulated under TNBS-induced colitis or oxidative stress. Pre-miR-122 or siRNA SBP1 (si-SBP1) treatment ameliorated acute TNBS-induced colitis and H2O2-induced oxidative stress. Cotreatment of pre-miR-122 and si-SBP1 enhanced these effects. Besides, pre-miR-122 and si-SBP1 obviously activated the p65NF-κB signaling by phosphorylation of IκBα. Bisulfite sequencing of the CpG islands in the promoter region of miR-122 showed that CpG methylation was significantly increased under oxidative stress. Treating cells with 5′-AZA which was well known as a DNA-demethylating agent significantly increased miR-122 expression. Our results suggest that oxidative stress-induced DNA methylation of miR-122 aggravates colitis targeting SELENBP1 partially by p65NF-κB signaling and may promote the progression of CD.

scholarly journals The Role of Sevenless in Drosophila R7 Photoreceptor Specification

2019 ◽  
Author(s):  
Andrew Tomlinson ◽  
Yannis Emmanuel Mavromatakis ◽  
Ronald Arias
Keyword(s):  
Receptor Tyrosine Kinase ◽  
Gene Function ◽  
Target Gene ◽  
Egf Receptor ◽  
Transmembrane Domain ◽  
Current Model ◽  
Intracellular Domain ◽  
Hydrophobic Domain ◽  
Pathway Activation

AbstractSevenless (Sev) is a Receptor Tyrosine Kinase (RTK) that is required for the specification of the Drosophila R7 photoreceptor. Other Drosophila photoreceptors are specified by the action of another RTK; the Drosophila EGF Receptor (DER). Why Sev is required specifically in the R7 precursor, and the exact role it plays in the cell’s fate assignment have long remained unclear. Notch (N) signaling plays many roles in R7 specification, one of which is to prevent DER activity from establishing the photoreceptor fate. Our current model of Sev function is that it hyperactivates the RTK pathway in the R7 precursor to overcome in the N-imposed block on photoreceptor specification. From this perspective DER and Sev are viewed as engaging the same transduction machinery, the only difference between them being the level of pathway activation that they induce. To test this model, we generated a Sev/DER chimera in which the intracellular domain of Sev is replaced with that of DER. This chimerical receptor acts indistinguishably from Sev itself; a result that is entirely consistent with the two RTKs sharing identical transduction abilities. A long-standing question in regard to Sev is the function of a hydrophobic domain some 60 amino acids from the initiating Methionine. If this represents a transmembrane domain, it would endow Sev with N-terminal intracellular sequences through which it could engage internal transduction pathways. However, we find that this domain acts as an internal signal peptide, and that there is no Sev N-terminal intracellular domain. phyllopod (phyl) is the target gene of the RTK pathway, and we show that R7 precursors are selectively lost when phyl gene function is mildly compromised, and that other photoreceptors are removed when the gene function is further reduced. This result adds a key piece of evidence for the hyperactivation of the RTK pathway in the R7 precursor. To facilitate the hyperactivation of the RTK pathway, Sev is expressed at high levels. However, when we express DER at the levels at which Sev is expressed, strong gain-of-function effects result, consistent with ligand-independent activation of the receptor. This highlights another key feature of Sev; that it is expressed at high levels yet remains strictly ligand dependent. Finally, we find that activated Sev can rescue R3/4 photoreceptors when their DER function is abrogated. These results are collectively consistent with Sev and DER activating the same transduction machinery, with Sev generating a pathway hyperactivation to overcome the N-imposed block to photoreceptor specification in R7 precursors.

scholarly journals RNA interference in schistosomes: machinery and methodology

Parasitology ◽  
2009 ◽  
Vol 137 (3) ◽  
pp. 485-495 ◽  
Author(s):  
GREICE KRAUTZ-PETERSON ◽  
RITA BHARDWAJ ◽  
ZAHRA FAGHIRI ◽  
CIBELE A. TARARAM ◽  
PATRICK J. SKELLY
Keyword(s):  
Rna Interference ◽  
Gene Function ◽  
Target Gene ◽  
Sirna Delivery ◽  
Review Of The Literature ◽  
Gene Suppression ◽  
Female Adult ◽  
Gene Encoding ◽  
C Elegans ◽  
Membrane Spanning

SUMMARYRNA interference (RNAi) is a potent gene silencing process that is playing an increasingly important role in investigations of gene function in schistosomes. Here we review what is known about the process in these parasites and provide an update on the methodology and machinery of RNAi. Data are presented to demonstrate that: (1) not all schistosome genes can be suppressed to the same extent, using the methods employed here; (2) while there is variation in the level of suppression achieved for one target gene (SmAP) in adult parasites, all individuals exhibit robust (>80%) suppression; (3) short interfering RNAs (siRNAs) can effect suppression when delivered by soaking (and not just via electroporation, as reported previously); (4) Male/female adult pairs need not be separated prior to siRNA delivery by electroporation for effective gene suppression in both genders and (5) electroporation of siRNAs in medium is as efficient as in commercial electroporation buffer. Regarding the machinery of RNAi in schistosomes, a homologue of the C. elegans multi-membrane spanning, RNA importing protein SID-1 is identified in silico. The gene encoding this protein contains 21 exons and spans over 50 kb to potentially encode a 115,556 Mr protein (SmSID-1). These analyses, and a review of the literature, permit us to derive and present here a draft of potential RNAi pathways in schistosomes.

scholarly journals Genome Editing with Crispr-Cas9 Systems: Basic Research and Clinical Applications

2017 ◽  
Vol 9 (1) ◽  
pp. 1
Author(s):  
Anna Meiliana ◽  
Nurrani Mustika Dewi ◽  
Andi Wijaya
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Gene Function ◽  
Target Gene ◽  
Large Range ◽  
Basic Research ◽  
Clinical Applications ◽  
Biological Research ◽  
Ethical Implication ◽  
Gene Modification

BACKGROUND: Recently established genome editing technologies will open new avenues for biological research and development. Human genome editing is a powerful tool which offers great scientific and therapeutic potential.CONTENT: Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein 9 (Cas9) technology is revolutionizing the gene function studies and possibly will give rise to an entirely new degree of therapeutics for a large range of diseases. Prompt advances in the CRISPR/Cas9 technology, as well as delivery modalities for gene therapy applications, are dismissing the barriers to the clinical translation of this technology. Many studies conducted showed promising results, but as current available technologies for evaluating off-target gene modification, several elements must be addressed to validate the safety of the CRISPR/Cas9 platform for clinical application, as the ethical implication as well.SUMMARY: The CRISPR/Cas9 system is a powerful genome editing technology with the potential to create a variety of novel therapeutics for a range of diseases, many of which are currently untreatable.KEYWORDS: genome editing, CRISPR-Cas, guideRNA, DSB, ZFNs, TALEN

scholarly journals CRISPR/CAS 9 Mediated Treatment for UTIs

2020 ◽  
Vol 6 (5) ◽  
pp. 82-94
Author(s):  
Sarika Chaturvedi ◽  
Jinny Tomar
Keyword(s):  
Escherichia Coli ◽  
Genetic Structure ◽  
Dna Sequences ◽  
Gene Function ◽  
Target Gene ◽  
Crispr Locus

“CRISPR" is short and used for "CRISPR-Cas9. CRISPR stands for clustered regularly interspaced short palindromic repeats. CRISPRs are specialized stretches of DNA. The protein Cas9 (or "CRISPR-associated") is an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA and can be used in conjunction with engineered CRISPR sequences to hunt down codes and slice into them like a molecular scalpel, allowing geneticists to cut out a target gene, either to remove it or replace it with a new sequence. Therefore it is a simple and powerful tool for editing genomes to easily alter DNA sequences and amend gene function. In 1987, The CRISPR locus was first identified in Escherichia coli and discovered when a genetic structure containing 5 highly homologous repeats of 29 nucleotides separated by 32-nucleotide spacers (Ishino Y 1987).

scholarly journals Development of a method for revertable CRISPR/Cas9-based mutagenesis in cell culture

2020 ◽  
Author(s):  
Jonathon Walsh ◽  
Jonathan Eggenschwiler
Keyword(s):  
Cell Culture ◽  
Gene Function ◽  
Target Gene ◽  
Culture Systems ◽  
Homology Directed Repair ◽  
Considerable Success ◽  
Targeted Mutation ◽  
Cell Culture Systems

AbstractCRISPR/Cas9 mutagenesis is a revolutionary tool for genetics in organismal and cell culture systems. One notable caveat with this system is the potential for phenotype-inducing off-target/background mutations. There has been considerable success in modifying the methodology to minimize these potential confounds. Here we have developed a tool to functionally demonstrate that a targeted mutation of interest is responsible for the phenotype observed. This approach creates revertable mutations in cell culture systems using CRISPR/Cas9-induced homology-directed repair (HDR) to insert a LoxP-flanked transcriptional stop sequence into an early intron of a target gene. This method has the potential to be used in multiplexed and inducible scenarios to restore gene function within a given experiment.

scholarly journals Gene knock-ins in Drosophila using homology-independent insertion of universal donor plasmids

2019 ◽  
Author(s):  
Justin A. Bosch ◽  
Ryan Colbeth ◽  
Jonathan Zirin ◽  
Norbert Perrimon
Keyword(s):  
Gene Function ◽  
Target Gene ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Germ Line ◽  
Insertion Site ◽  
End Joining ◽  
Endogenous Proteins ◽  
Non Homologous End Joining ◽  
Universal Donor

AbstractTargeted genomic knock-ins are a valuable tool to probe gene function. However, knock-in methods involving homology-directed repair (HDR) can be laborious. Here, we adapt the mammalian CRISPaint homology-independent knock-in method for Drosophila melanogaster, which uses CRISPR/Cas9 and non-homologous end joining (NHEJ) to insert universal donor plasmids into the genome. This method is a simple and fast alternative to HDR for certain strategies such as C-terminal tagging and gene disruption. Using this method in cultured S2R+ cells, we efficiently tagged four endogenous proteins with the bright fluorescent protein mNeonGreen, thereby demonstrating that an existing collection of CRISPaint universal donor plasmids is compatible with insect cells. In addition, we inserted the transgenesis marker 3xP3-RFP into seven genes in the fly germ line, producing heritable loss of function alleles that were isolated by simple fluorescence screening. Unlike in cultured cells, indels always occurred at the genomic insertion site, which prevents predictably matching the insert coding frame to the target gene. Despite this effect, we were able to isolate T2A-Gal4 insertions in four genes that serve as in vivo expression reporters. Finally, we apply this fast knock-in method to uncharacterized small open reading frame (smORF) genes. Therefore, homology-independent insertion is a useful genome editing technique in Drosophila that will better enable researchers to dissect gene function.Article summaryWe report a fast and simple genomic knock-in method in Drosophila to insert large DNA elements into any target gene. Using CRISPR-Cas9 and non-homologous end joining (NHEJ), an entire donor plasmid is inserted into the genome without the need for homology arms. We demonstrate its usefulness in cultured cells to fluorescently tag endogenous proteins and in the fly germ line to generate heritable insertions that disrupt gene function and can act as expression reporters.

scholarly journals TUTase mediated site-directed access to clickable chromatin employing CRISPR-dCas9

2019 ◽  
Author(s):  
Jerrin Thomas George ◽  
Mohd. Azhar ◽  
Meghali Aich ◽  
Dipanjali Sinha ◽  
Uddhav B. Ambi ◽  
...  
Keyword(s):  
Minimally Invasive ◽  
Small Molecules ◽  
Click Chemistry ◽  
Gene Function ◽  
Molecular Basis ◽  
Target Gene ◽  
Target Genes ◽  
Click Reactions ◽  
Simplified Solution ◽  
Modular Technology

AbstractLocus-specific interrogation of the genome using programmable CRISPR-based technologies is tremendously useful in dissecting the molecular basis of target gene function and modulating its downstream output. Although these tools are widely utilized in recruiting genetically encoded functional proteins, display of small molecules using this technique is not well developed due to inadequate labeling technologies. Here, we report the development of a modular technology, sgRNA-Click (sgR-CLK), which harnesses the power of bioorthogonal click chemistry for remodeling CRISPR to display synthetic molecules on target genes. A terminal uridylyl transferase (TUTase) was repurposed to construct an sgRNA containing multiple minimally invasive bioorthogonal clickable handles, which served as a Trojan horse on CRISPR-dCas9 system to guide synthetic tags site-specifically on chromatin employing copper-catalyzed or strain-promoted click reactions. Our results demonstrate that sgR-CLK could provide a simplified solution for site-directed display of small molecules to study as well as modulate the function of gene targets.

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