scholarly journals CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean

2018 ◽  
Vol 19 (12) ◽  
pp. 3835 ◽  
Author(s):  
Yupeng Cai ◽  
Li Chen ◽  
Shi Sun ◽  
Cunxiang Wu ◽  
Weiwei Yao ◽  
...  
Keyword(s):  
Target Genes ◽  
Site Directed Mutagenesis ◽  
Future Research ◽  
Dna Fragments ◽  
Cleavage Sites ◽  
Chromosome Engineering ◽  
Efficient System ◽  
Guide Rnas ◽  
Glycine Max L ◽  
Deletion Frequency

At present, the application of CRISPR/Cas9 in soybean (Glycine max (L.) Merr.) has been mainly focused on knocking out target genes, and most site-directed mutagenesis has occurred at single cleavage sites and resulted in short deletions and/or insertions. However, the use of multiple guide RNAs for complex genome editing, especially the deletion of large DNA fragments in soybean, has not been systematically explored. In this study, we employed CRISPR/Cas9 technology to specifically induce targeted deletions of DNA fragments in GmFT2a (Glyma16g26660) and GmFT5a (Glyma16g04830) in soybean using a dual-sgRNA/Cas9 design. We achieved a deletion frequency of 15.6% for target fragments ranging from 599 to 1618 bp in GmFT2a. We also achieved deletion frequencies of 12.1% for target fragments exceeding 4.5 kb in GmFT2a and 15.8% for target fragments ranging from 1069 to 1161 bp in GmFT5a. In addition, we demonstrated that these CRISPR/Cas9-induced large fragment deletions can be inherited. The T2 ‘transgene-free’ homozygous ft2a mutants with a 1618 bp deletion exhibited the late-flowering phenotype. In this study, we developed an efficient system for deleting large fragments in soybean using CRISPR/Cas9; this system could benefit future research on gene function and improve agriculture via chromosome engineering or customized genetic breeding in soybean.

An efficient system for markerless gene replacement applicable in a wide variety of enterobacterial species

2007 ◽  
Vol 53 (1) ◽  
pp. 56-62 ◽  
Author(s):  
A P White ◽  
E Allen-Vercoe ◽  
B W Jones ◽  
R DeVinney ◽  
W W Kay ◽  
...  
Keyword(s):  
Target Genes ◽  
Bacterial Species ◽  
Gene Replacement ◽  
Site Directed Mutagenesis ◽  
Temperature Sensitive ◽  
Chromosomal Dna ◽  
Exchange Method ◽  
Efficient System ◽  
Allelic Exchange ◽  
Genetic Manipulations

We describe an improved allelic-exchange method for generating unmarked mutations and chromosomal DNA alterations in enterobacterial species. Initially developed for use in Salmonella enterica, we have refined the method in terms of time, simplicity, and efficiency. We have extended its use into related bacterial species that are more recalcitrant to genetic manipulations, including enterohemorrhagic and enteropathogenic Escherichia coli and Vibrio parahaemolyticus. Data from over 50 experiments are presented including gene inactivations, site-directed mutagenesis, and promoter exchanges. In each case, desired mutations were identified by polymerase chain reaction screening typically from as few as 10–20 colonies up to a maximum of 300 colonies. The method does not require antibiotic nor nutritional markers in target genes and works efficiently in wild-type strains, obviating the need for specialized hosts or genetic systems. The use is simple, requiring basic laboratory materials, and represents an alternative to existing methods for gene manipulation in the Enterobacteriaceae.Key words: allelic exchange, temperature-sensitive plasmids.

scholarly journals New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing

2020 ◽  
Vol 22 (1) ◽  
pp. 319
Author(s):  
Jaiana Malabarba ◽  
Elisabeth Chevreau ◽  
Nicolas Dousset ◽  
Florian Veillet ◽  
Julie Moizan ◽  
...  
Keyword(s):  
Target Genes ◽  
Cytidine Deaminase ◽  
Acetolactate Synthase ◽  
Nucleotide Substitutions ◽  
Discrete Variation ◽  
Adventitious Regeneration ◽  
Base Editing ◽  
Perennial Plants ◽  
Guide Rnas ◽  
Albino Phenotype

Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the phytoene desaturase (PDS) gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for “regenerated T0” lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: acetolactate synthase—ALS (conferring resistance to chlorsulfuron) and PDS. The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing ALS and PDS genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants.

scholarly journals Genome-wide miRNA profiling of villus and decidua of recurrent spontaneous abortion patients

Reproduction ◽  
2014 ◽  
Vol 148 (1) ◽  
pp. 33-41 ◽  
Author(s):  
Fulu Dong ◽  
Yuan Zhang ◽  
Fei Xia ◽  
Yi Yang ◽  
Sidong Xiong ◽  
...  
Keyword(s):  
Spontaneous Abortion ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Expression Profiles ◽  
Normal Pregnancy ◽  
Recurrent Spontaneous Abortion ◽  
Future Research ◽  
Rna Molecules ◽  
Non Coding Rna ◽  
Transcriptional Gene Regulation

MicroRNAs (miRNAs) are non-coding RNA molecules of about 22 nucleotides that involved in post-transcriptional gene regulation. Evidence indicates that miRNAs play essential roles in endometriosis, pre-eclampsia, infertility and other reproductive system diseases. However, whether miRNAs are involved in recurrent spontaneous abortion (RSA) is unclear. In this work, we analysed the miRNA expression profiles in six pairs of villus or decidua from RSA patients and normal pregnancy (NP) women using a human miRNA microarray. Some of the chip results were confirmed by RT-qPCR. In the villi of RSA patients, expression of hsa-miR-184, hsa-miR-187 and hsa-miR-125b-2 was significantly higher, while expression of hsa-miR-520f, hsa-miR-3175 and hsa-miR-4672 was significantly lower, comparing with those of NP control. As well, a total of five miRNAs (hsa-miR-517c, hsa-miR-519a-1, hsa-miR-522, hsa-miR-520h and hsa-miR-184) were upregulated in the decidua of RSA patients. The target genes of these differentially expressed miRNAs were predicted by miRWalk, and we speculate a network of miRNA regulating RSA by target genes function on adhesion, apoptosis and angiogenesis. Our study may help clarify the molecular mechanisms which are involved in the progression of RSA, and provide a reference for future research.

scholarly journals N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

2015 ◽  
Vol 82 (4) ◽  
pp. 1004-1014 ◽  
Author(s):  
Canfang Niu ◽  
Huiying Luo ◽  
Pengjun Shi ◽  
Huoqing Huang ◽  
Yaru Wang ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Biochemical Characterization ◽  
Animal Feed ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Acidic Ph ◽  
Cleavage Sites ◽  
Ph Optimum ◽  
Content Type ◽  
Histidine Acid Phosphatase

ABSTRACTN-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases fromYersinia kristensenii(YkAPPA) andYersinia rohdei(YrAPPA), each having anN-glycosylation motif, and one pepsin-sensitive HAP phytase fromYersinia enterocolitica(YeAPPA) that lacked anN-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering theN-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed inPichia pastorisfor biochemical characterization. Compared with those of theN-glycosylation site deletion mutants andN-deglycosylated enzymes, allN-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of theN-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of theN-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced inEscherichia colibut had no effect on the pepsin resistance ofN-glycosylated enzymes produced inP. pastoris. Thus,N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation ofN-glycosylation, for improvement of phytase properties for use in animal feed.

scholarly journals Integration and Visualization of Regulatory Elements and Variations of the EPAS1 Gene in Human

Genes ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1793
Author(s):  
Aleša Kristan ◽  
Nataša Debeljak ◽  
Tanja Kunej
Keyword(s):  
Target Genes ◽  
Regulatory Elements ◽  
Future Research ◽  
Protein Abundance ◽  
Interacting Proteins ◽  
Pas Domain ◽  
Expression Stability ◽  
Post Translational Modifications ◽  
Oxygen Homeostasis ◽  
Cancer Types

Endothelial PAS domain-containing protein 1 (EPAS1), also HIF2α, is an alpha subunit of hypoxia-inducible transcription factor (HIF), which mediates cellular and systemic response to hypoxia. EPAS1 has an important role in the transcription of many hypoxia-responsive genes, however, it has been less researched than HIF1α. The aim of this study was to integrate an increasing number of data on EPAS1 into a map of diverse OMICs elements. Publications, databases, and bioinformatics tools were examined, including Ensembl, MethPrimer, STRING, miRTarBase, COSMIC, and LOVD. The EPAS1 expression, stability, and activity are tightly regulated on several OMICs levels to maintain complex oxygen homeostasis. In the integrative EPAS1 map we included: 31 promoter-binding proteins, 13 interacting miRNAs and one lncRNA, and 16 post-translational modifications regulating EPAS1 protein abundance. EPAS1 has been associated with various cancer types and other diseases. The development of neuroendocrine tumors and erythrocytosis was shown to be associated with 11 somatic and 20 germline variants. The integrative map also includes 12 EPAS1 target genes and 27 interacting proteins. The study introduced the first integrative map of diverse genomics, transcriptomics, proteomics, regulomics, and interactomics data associated with EPAS1, to enable a better understanding of EPAS1 activity and regulation and support future research.

scholarly journals Identification of key genes and associated pathways in neuroendocrine tumors through bioinformatics analysis and predictions of small drug molecules

2020 ◽  
Author(s):  
Praveenkumar Devarbhavi ◽  
Basavaraj Vastrad ◽  
Anandkumar Tengli ◽  
Chanabasayya Vastrad ◽  
Iranna Kotturshetti
Keyword(s):  
Drug Target ◽  
Target Gene ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Regulatory Elements ◽  
Future Research ◽  
High Morbidity ◽  
Hub Genes ◽  
Go Enrichment ◽  
Significant Difference

AbstractNeuroendocrine tumor (NET) is one of malignant cancer and is identified with high morbidity and mortality rates around the world. With indigent clinical outcomes, potential biomarkers for diagnosis, prognosis and drug target are crucial to explore. The aim of this study is to examine the gene expression module of NET and to identify potential diagnostic and prognostic biomarkers as well as to find out new drug target. The differentially expressed genes (DEGs) identified from GSE65286 dataset was used for pathway enrichment analyses and gene ontology (GO) enrichment analyses and protein - protein interaction (PPI) analysis and module analysis. Moreover, miRNAs and transcription factors (TFs) that regulated the up and down regulated genes were predicted. Furthermore, validation of hub genes was performed. Finally, molecular docking studies were performed. DEGs were identified, including 453 down regulated and 459 up regulated genes. Pathway and GO enrichment analysis revealed that DEGs were enriched in sucrose degradation, creatine biosynthesis, anion transport and modulation of chemical synaptic transmission. Important hub genes and target genes were identified through PPI network, modules, target gene - miRNA network and target gene - TF network. Finally, survival analyses, receiver operating characteristic (ROC) curve and RT-PCR validated the significant difference of ATP1A1, LGALS3, LDHA, SYK, VDR, OBSL1, KRT40, WWOX, NINL and PPP2R2B between metastatic NET and normal controls. In conclusion, the DEGs and hub genes with their regulatory elements identified in this study will help us understand the molecular mechanisms underlying NET and provide candidate targets for future research.

MicroRNAs: role in cardiovascular biology and disease

2008 ◽  
Vol 114 (12) ◽  
pp. 699-706 ◽  
Author(s):  
Chunxiang Zhang
Keyword(s):  
Cardiovascular Diseases ◽  
Target Genes ◽  
Future Research ◽  
Cardiovascular Development ◽  
Biological Functions ◽  
Lesion Formation ◽  
Regulate Gene Expression ◽  
Proliferation And Apoptosis ◽  
Cardiovascular Biology ◽  
Non Coding Rnas

miRNAs (microRNAs) comprise a novel class of endogenous, small, non-coding RNAs that negatively regulate gene expression via degradation or translational inhibition of their target mRNAs. Recent studies have demonstrated that miRNAs are highly expressed in the cardiovascular system. Although we are currently in the initial stages of understanding how this novel class of gene regulators is involved in cardiovascular biological functions, a growing body of exciting evidence suggests that miRNAs are important regulators of cardiovascular cell differentiation, growth, proliferation and apoptosis. Moreover, miRNAs are key modulators of both cardiovascular development and angiogenesis. Consequently, dysregulation of miRNA function may lead to cardiovascular diseases. Indeed, several recent reports have demonstrated that miRNAs are aberrantly expressed in diseased hearts and vessels. Modulating these aberrantly expressed miRNAs has significant effects on cardiac hypertrophy, vascular neointimal lesion formation and cardiac arrhythmias. Identifying the roles of miRNAs and their target genes and signalling pathways in cardiovascular disease will be critical for future research. miRNAs may represent a new layer of regulators for cardiovascular biology and a novel class of therapeutic targets for cardiovascular diseases.

scholarly journals The methylated-DNA binding protein MBD2 enhances NGFI-A (egr-1)-mediated transcriptional activation of the glucocorticoid receptor

2014 ◽  
Vol 369 (1652) ◽  
pp. 20130513 ◽  
Author(s):  
Ian C. G. Weaver ◽  
Ian C. Hellstrom ◽  
Shelley E. Brown ◽  
Stephen D. Andrews ◽  
Sergiy Dymov ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Protein A ◽  
Site Directed Mutagenesis ◽  
Signalling Pathways ◽  
Hek 293 ◽  
293 Cells ◽  
Exon 1 ◽  
Inducible Protein

Variations in maternal care in the rat influence the epigenetic state and transcriptional activity of glucocorticoid receptor (GR) gene in the hippocampus. The mechanisms underlying this maternal effect remained to be defined, including the nature of the relevant maternally regulated intracellular signalling pathways. We show here that increased maternal licking/grooming (LG), which stably enhances hippocampal GR expression, paradoxically increases hippocampal expression of the methyl-CpG binding domain protein-2 (MBD2) and MBD2 binding to the exon 1 7 GR promoter. Knockdown experiments of MBD2 in hippocampal primary cell culture show that MBD2 is required for activation of exon 1 7 GR promoter. Ectopic co-expression of nerve growth factor-inducible protein A (NGFI-A) with MBD2 in HEK 293 cells with site-directed mutagenesis of the NGFI-A response element within the methylated exon 1 7 GR promoter supports the hypothesis that MBD2 collaborates with NGFI-A in binding and activation of this promoter. These data suggest a possible mechanism linking signalling pathways, which are activated by behavioural stimuli and activation of target genes.

scholarly journals The transcriptional activator hepatocyte nuclear factor 6 regulates liver gene expression.

1996 ◽  
Vol 16 (11) ◽  
pp. 6273-6284 ◽  
Author(s):  
U Samadani ◽  
R H Costa
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Dna Binding ◽  
Target Genes ◽  
Site Directed Mutagenesis ◽  
Epithelial Cell Line ◽  
Homeodomain Protein ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Fatty Acid Binding

The hepatocyte nuclear factor 3(alpha) (HNF-3(alpha)), -3(beta), and -3(gamma) proteins share homology in the winged-helix/fork head DNA binding domain and mediate hepatocyte-enriched transcription of numerous genes whose expression is necessary for organ function. In this work, we identify a liver-enriched transcription factor, HNF-6, which recognizes the -138 to -126 region of the HNF-3(beta) promoter and binds the original HNF-3 site of the transthyretin promoter (-94 to -106). We show that HNF-6 and HNF-3 possess different DNA binding specificities by competition and methylation interference studies and are immunologically distinct. Site-directed mutagenesis of the HNF-6 sites in the HNF-3(beta) and transthyretin promoters diminishes reporter gene expression, suggesting that HNF-6 activates transcription of these promoters. Using the HNF-6 binding sequence DHWATTGAYTWWD (where W = A or T, Y = T or C, H is not G, and D is not C) determined by sequence comparison and methylation interference, we predicted that HNF-6 will bind to 22 additional hepatocyte-enriched genes. Of these potential target genes, we selected seven of the HNF-6 binding sequences and demonstrated that they bind the HNF-6 protein. These include promoter sequences from alpha-2 urinary globulin, alpha-1 antitrypsin, cytochrome P-450 2C13, L-type 6-phosphofructo-2-kinase, mouse major urinary protein, tryptophan oxygenase, and alpha-fetoprotein genes. HNF-6 binding activity was also found in the intestinal epithelial cell line HT29, and potential HNF-6 binding sites were present in intestinal sucrase isomaltase, cdx-2 homeodomain protein, and intestinal fatty acid binding protein promoter regions. These studies suggest that HNF-6 may regulate hepatocyte-specific genes and may play a role in epithelial cell differentiation of gut endoderm via regulation of HNF-3(beta).

338 BACTERIAL ARTIFICIAL CHROMOSOME (BAC) VECTORS FACILITATE EFFICIENT GENE TARGETTING IN KIDNEY CELLS OF PIG

2011 ◽  
Vol 23 (1) ◽  
pp. 264
Author(s):  
K. Wallner ◽  
A. Wuensch ◽  
K. Burkhardt ◽  
M. Kurome ◽  
B. Kessler ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Biomedical Research ◽  
Nuclear Transfer ◽  
Target Genes ◽  
Embryonic Stem ◽  
Artificial Chromosome ◽  
Site Directed Mutagenesis ◽  
Kidney Cells ◽  
Fetal Fibroblasts ◽  
Dmd Gene

Site-directed mutagenesis provided a powerful tool for studying gene functions in mice, but the lack of embryonic stem cells in other species limited the application of this technology to other species. Various attempts using negative selection, viral vectors, or other auxiliary means promoted specific projects but did not provide methods for routine experiments. Here, we describe a novel approach that enabled the site-directed modification of 3 different porcine genes relevant for biomedical research. Three main technologies were combined to achieve these goals: bacterial artificial chromosome (BAC) vectors, somatic cell transfection, and nuclear transfer (SCNT). BAC vectors contain large genomic regions in bacterial plasmids. They are superior to conventional targeting tools, as they provide extended regions of homology of several kilobases. Novel recombination tools using bacterial enzymes enable the modification of any DNA region of interest and thus allow the introduction of desired mutations into BACs. After verification of the wt-BAC sequence, it was altered by using modification vectors carrying the desired mutation. The modified BAC vectors are linearized and transfected after verification into primary kidney cell lines, and cells are selected for integration of the vectors. Kidney cells provide both good proliferation and high targeting rates, and thus improve the efficiency compared to fetal fibroblasts. Singularized clones are screened for the replacement of wild-type targeting loci by quantitative PCR. Targeted clones are used for SCNT and transfer of the resulting embryos into synchronized gilts. We have evaluated this technology by the modification of the porcine CFTR, GGTA1, and DMD genes. All 3 genes are relevant for biomedical research, as mutations in CFTR are causative for cystic fibrosis, the knockout of GGTA1 is essential for overcoming hyperacute rejection in xenotransplantation, and various deletions in the DMD gene are responsible for Duchenne muscular dystrophy. Gaining 13 targeted clones out of 1152 for CFTR, 9 out of 306 for GGTA1 and 6 out of 203 for DMD, we obtained efficiencies higher than 1% for each of the target genes. The power of our approach is underlined by the fact that CFTR and DMD are loci that are thought to be difficult to manipulate. The viability of targeted kidney cells and their suitability for nuclear transfer is accentuated by the pregnancy rates (2 out of 3) and the delivery of 4 to 10 piglets or fetuses in the case of CFTR and GGTA1. The heterozygous fetuses or piglets are verified by qPCR. In the case of the X-chromosomal DMD gene, we have generated the first full knockout by transfecting male cells. Pregnancies of a successfully targeted clone are under way. Thus, we consider the combination of modified BAC vectors, transfection of kidney cells, and nuclear transfer to be a technology with the potential for routine production of site-directed mutations. Supported by the Mukoviszidose e.V. and the Bayerische Forschungsstiftung.

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