scholarly journals The Functional Association of ACQOS/VICTR with Salt Stress Resistance in Arabidopsis thaliana Was Confirmed by CRISPR-Mediated Mutagenesis

2021 ◽  
Vol 22 (21) ◽  
pp. 11389
Author(s):  
Sang-Tae Kim ◽  
Minkyung Choi ◽  
Su-Ji Bae ◽  
Jin-Soo Kim
Keyword(s):  
Salt Stress ◽  
Target Genes ◽  
Gene Knockout ◽  
Salt Resistance ◽  
Knock Out ◽  
Chlorophyll Contents ◽  
Guide Rnas ◽  
Osmotic Tolerance ◽  
Multiple Genes ◽  
Acclimation Period

Clustered regularly interspaced palindromic repeat (CRISPR)-mediated mutagenesis has become an important tool in plant research, enabling the characterization of genes via gene knock-out. CRISPR genome editing tools can be applied to generate multi-gene knockout lines. Typically, multiple single-stranded, single guide RNAs (gRNAs) must be expressed in an organism to target multiple genes simultaneously; however, a single gRNA can target multiple genes if the target genes share similar sequences. A gene cluster comprising ACQUIRED OSMOTOLERANCE (ACQOS; AT5G46520) and neighboring nucleotide-binding leucine-rich repeats (NLRs; AT5G46510) is associated with osmotic tolerance. To investigate the role of ACQOS and the tandemly arranged NLR in osmotic tolerance, we introduced small insertion/deletion mutations into two target genes using a single gRNA and obtained transformant plant lines with three different combinations of mutant alleles. We then tested our mutant lines for osmotic tolerance after a salt-stress acclimation period by determining the chlorophyll contents of the mutant seedlings. Our results strongly suggest that ACQOS is directly associated with salt resistance, while the neighboring NLR is not. Here, we confirmed previous findings suggesting the involvement of ACQOS in salt tolerance and demonstrated the usefulness of CRISPR-mediated mutagenesis in validating the functions of genes in a single genetic background.

scholarly journals Comparative transcriptome analysis of sweet sorghum provides insights into new lncRNAs acting as ceRNAs during salt responses

2019 ◽  
Author(s):  
Xi Sun ◽  
Hongxiang Zheng ◽  
Jinlu Li ◽  
Na Sui
Keyword(s):  
Salt Stress ◽  
Sweet Sorghum ◽  
Target Genes ◽  
Salt Resistance ◽  
Rna Seq ◽  
Salt Stress Response ◽  
Rna Transcripts ◽  
Competitive Endogenous Rnas ◽  
Sensitive Line ◽  
Signal Transduction Proteins

Abstract LncRNAs can act as competitive endogenous RNAs (ceRNAs) to competitively bind miRNAs, thereby indirectly regulating the transcription levels of other RNA transcripts to confer resistance to plants. But how specific ceRNAs respond to salt stress in sweet sorghum is still unclear. In this study, 126 and 133 differentially expressed lncRNAs were identified in salt-tolerant sweet sorghum (M-81E) and a salt-sensitive line (Roma) by high-throughput RNA-seq, respectively. And five new lncRNAs were identified in M-81E and Roma after salt stress treatment, lncRNA13472, lncRNA11310, lncRNA2846, lncRNA26929 and lncRNA14798 acted as ceRNAs to regulate the expression of target genes related to salt resistance by binding the five miRNAs sbi-MIR169b-p3, sbi-MIR5567-p3-2ss16CT17TC, sbi-MIR5567-p5-2, sbi-MIR5567-p5-2ss17CT18TC and PC-3p-270284-34, respectively. The target genes mainly included proton pump proteins, transport proteinsantioxidants, signal transduction proteins and transcription factors. However M-81E had more complex ceRNAs network than in Roma, which might be related with its different salt tolerance. In summary, this study identified a new ceRNA network within the transcriptome and revealed the effect of lncRNAs in the salt stress response.

scholarly journals Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the FAD2 and FATB Multigene in Soybean

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2542
Author(s):  
Won-Nyeong Kim ◽  
Hye-Jeong Kim ◽  
Young-Soo Chung ◽  
Hyun-Uk Kim
Keyword(s):  
Reverse Genetics ◽  
Restriction Enzymes ◽  
Golden Gate ◽  
Knock Out ◽  
Guide Rnas ◽  
Genetics Research ◽  
Multiple Genes ◽  
Cas9 Protein ◽  
Cloning Method ◽  
Golden Gate Cloning

CRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 protein to the target site. In this protocol, the Golden Gate cloning method was used to generate multiple sgRNAs in the Cas9 vector. The vectors used were pHEE401E_UBQ_Bar and pBAtC_tRNA, which employ a one-promoter/one-sgRNA and a polycistronic-tRNA-gRNA strategy, respectively. Golden Gate cloning was performed with type IIS restriction enzymes to generate gRNA polymers for vector inserts. Four sgRNAs containing the pHEE401E_UBQ_Bar vector and four to six sgRNAs containing the pBAtC_tRNA vector were constructed. In practice, we constructed multiple sgRNAs targeting multiple genes of FAD2 and FATB in soybean using this protocol. These three vectors were transformed into soybeans using the Agrobacterium-mediated method. Using deep sequencing, we confirmed that the T0 generation transgenic soybean was edited at various indel ratios in the predicted target regions of the FAD2 and FATB multigenes. This protocol is a specific guide that allows researchers to easily follow the cloning of multiple sgRNAs into commonly used CRISPR/Cas9 vectors for plants.

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.

NFATc3 regulates the transcription of genes involved in T-cell activation and angiogenesis

Blood ◽  
2011 ◽  
Vol 118 (3) ◽  
pp. 795-803 ◽  
Author(s):  
Katia Urso ◽  
Arantzazu Alfranca ◽  
Sara Martínez-Martínez ◽  
Amelia Escolano ◽  
Inmaculada Ortega ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Target Genes ◽  
Gene Knockout ◽  
Activated T Cells ◽  
Knock Down ◽  
And Function

Abstract The nuclear factor of activated T cells (NFAT) family of transcription factors plays important roles in many biologic processes, including the development and function of the immune and vascular systems. Cells usually express more than one NFAT member, raising the question of whether NFATs play overlapping roles or if each member has selective functions. Using mRNA knock-down, we show that NFATc3 is specifically required for IL2 and cyclooxygenase-2 (COX2) gene expression in transformed and primary T cells and for T-cell proliferation. We also show that NFATc3 regulates COX2 in endothelial cells, where it is required for COX2, dependent migration and angiogenesis in vivo. These results indicate that individual NFAT members mediate specific functions through the differential regulation of the transcription of target genes. These effects, observed on short-term suppression by mRNA knock-down, are likely to have been masked by compensatory effects in gene-knockout studies.

scholarly journals Interaction of SOS2 with Nucleoside Diphosphate Kinase 2 and Catalases Reveals a Point of Connection between Salt Stress and H2O2 Signaling in Arabidopsis thaliana

2007 ◽  
Vol 27 (22) ◽  
pp. 7771-7780 ◽  
Author(s):  
Paul E. Verslues ◽  
Giorgia Batelli ◽  
Stefania Grillo ◽  
Fernanda Agius ◽  
Yong-Sig Kim ◽  
...  
Keyword(s):  
Salt Stress ◽  
Stress Response ◽  
Stress Responses ◽  
Double Mutant ◽  
Nucleoside Diphosphate Kinase ◽  
Salt Resistance ◽  
Interacting Proteins ◽  
Salt Stress Response ◽  
Oxygen Signaling ◽  
Nucleoside Diphosphate Kinase 2

ABSTRACT SOS2, a class 3 sucrose-nonfermenting 1-related kinase, has emerged as an important mediator of salt stress response and stress signaling through its interactions with proteins involved in membrane transport and in regulation of stress responses. We have identified additional SOS2-interacting proteins that suggest a connection between SOS2 and reactive oxygen signaling. SOS2 was found to interact with the H2O2 signaling protein nucleoside diphosphate kinase 2 (NDPK2) and to inhibit its autophosphorylation activity. A sos2-2 ndpk2 double mutant was more salt sensitive than a sos2-2 single mutant, suggesting that NDPK2 and H2O2 are involved in salt resistance. However, the double mutant did not hyperaccumulate H2O2 in response to salt stress, suggesting that it is altered signaling rather than H2O2 toxicity alone that is responsible for the increased salt sensitivity of the sos2-2 ndpk2 double mutant. SOS2 was also found to interact with catalase 2 (CAT2) and CAT3, further connecting SOS2 to H2O2 metabolism and signaling. The interaction of SOS2 with both NDPK2 and CATs reveals a point of cross talk between salt stress response and other signaling factors including H2O2.

scholarly journals A CRISPR/Cas9 genome editing pipeline in the EndoC-βH1 cell line to study genes implicated in beta cell function

2019 ◽  
Vol 4 ◽  
pp. 150 ◽  
Author(s):  
Antje K. Grotz ◽  
Fernando Abaitua ◽  
Elena Navarro-Guerrero ◽  
Benoit Hastoy ◽  
Daniel Ebner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Beta Cell ◽  
Er Stress ◽  
Cell Line ◽  
Cell Lines ◽  
Target Genes ◽  
Gene Knockout ◽  
Mrna Levels ◽  
Beta Cell Line ◽  
Human Beta Cell

Type 2 diabetes (T2D) is a global pandemic with a strong genetic component, but most causal genes influencing the disease risk remain unknown. It is clear, however, that the pancreatic beta cell is central to T2D pathogenesis. In vitro gene-knockout (KO) models to study T2D risk genes have so far focused on rodent beta cells. However, there are important structural and functional differences between rodent and human beta cell lines. With that in mind, we have developed a robust pipeline to create a stable CRISPR/Cas9 KO in an authentic human beta cell line (EndoC-βH1). The KO pipeline consists of a dual lentiviral sgRNA strategy and we targeted three genes (INS, IDE, PAM) as a proof of concept. We achieved a significant reduction in mRNA levels and complete protein depletion of all target genes. Using this dual sgRNA strategy, up to 94 kb DNA were cut out of the target genes and the editing efficiency of each sgRNA exceeded >87.5%. Sequencing of off-targets showed no unspecific editing. Most importantly, the pipeline did not affect the glucose-responsive insulin secretion of the cells. Interestingly, comparison of KO cell lines for NEUROD1 and SLC30A8 with siRNA-mediated knockdown (KD) approaches demonstrate phenotypic differences. NEUROD1-KO cells were not viable and displayed elevated markers for ER stress and apoptosis. NEUROD1-KD, however, only had a modest elevation, by 34%, in the pro-apoptotic transcription factor CHOP and a gene expression profile indicative of chronic ER stress without evidence of elevated cell death. On the other hand, SLC30A8-KO cells demonstrated no reduction in KATP channel gene expression in contrast to siRNA silencing. Overall, this strategy to efficiently create stable KO in the human beta cell line EndoC-βH1 will allow for a better understanding of genes involved in beta cell dysfunction, their underlying functional mechanisms and T2D pathogenesis.

scholarly journals Influence of salt stress on plants of poplar clone "INRA 353-38" and willow clone "Zhytomyrska-1" in in vitro culture

2020 ◽  
Vol 83 (4) ◽  
pp. 43-49
Author(s):  
Yu. Khoma ◽  
L. Khudolieieva ◽  
N. Kutsokon
Keyword(s):  
Salt Stress ◽  
Sodium Chloride ◽  
In Vitro Culture ◽  
Growth Parameters ◽  
Hybrid Poplar ◽  
Salt Resistance ◽  
Soil Salinization ◽  
In Vitro Cultivation ◽  
Abiotic Factor

Soil salinization is an important abiotic factor negatively affecting plant growth, development and productivity. Fast-growing poplar and willow trees are important plants for bioenergy production demonstrating varying degrees of adaptation to different habitats. The study of salt resistance in different clones of poplars and willows will reveal genotypes that can be planted in saline soils for producing biomass for the bioenergy industry. Therefore, the aim of the study was to investigate the effects of salt stress on poplar plants of clone 'INRA 353-38' (Populus tremula × P. tremuloides) and willow clone 'Zhytomyrska – 1' (Salix sp.) under in vitro culture. For this purpose the plants were cultivated on MS nutrient medium with the addition of sodium chloride in concentrations 25 mM, 50 mM and 100 mM. The control plants were grown on the sodium chloridefree medium. The plant status (with a 4-score scale), the intensity of their growth (by shoot length) and rooting capacity (by the number of roots) were assessed on the 10th and the 30th day of cultivation. The results obtained indicate a high level of sensitivity to sodium chloride of both studied clones under in vitro cultivation. But the willow 'Zhytomyrska – 1' had a higher sensitivity to salt stress comparing to hybrid polar 'ІNRA 353-38' since growth parameters of willow were significantly decreased even under the concentration of sodium chloride 50 mM, and in the case of short term influence (10 days) of the highest concentration of sodium chloride (100 mM) all willow plants terminated their growth and quickly died. The growth parameters of hybrid poplar were declined within a month, mainly under the highest concentration of sodium chloride, but even under such conditions some part of the shoots were able to survive.

Effects of expression of p53 and Gadd45 on osmotic tolerance of renal inner medullary cells

2006 ◽  
Vol 291 (2) ◽  
pp. F341-F349 ◽  
Author(s):  
Qi Cai ◽  
Natalia I. Dmitrieva ◽  
Joan D. Ferraris ◽  
Luis F. Michea ◽  
Jesus M. Salvador ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Collecting Duct ◽  
Wild Type ◽  
Growth And Survival ◽  
Osmotic Tolerance ◽  
Rate Of Increase ◽  
Gene Knockout Mice ◽  
Collecting Duct Cells ◽  
Urinary Concentrating Ability

The response of renal inner medullary (IM) collecting duct cells (mIMCD3) to high NaCl involves increased expression of Gadd45 and p53, both of which have important effects on growth and survival of the cells. However, mIMCD3 cells, being immortalized by SV40, proliferate rapidly, which is known to sensitize cells to high NaCl, whereas IM cells in situ proliferate very slowly and survive much higher levels of NaCl. In the present studies, we have examined the importance of Gadd45 and p53 for survival of normal IM cells in their usual high-NaCl environment by using more slowly proliferating second-passage mouse inner medullary epithelial (p2mIME) cells and comparing cells from wild-type and gene knockout mice. Acutely elevating NaCl (and/or urea) reduces Gadd45a, but increases Gadd45b and Gadd45g mRNA, depending on the mix of NaCl and urea and the rate of increase of osmolality. Nevertheless, p2mIME cells from Gadd45b−/−, Gadd45g−/−, and Gadd45bg−/− mice survive elevation of NaCl (or urea) essentially the same as do wild-type cells. p53−/− Cells do not tolerate as high a concentration of NaCl (or urea) as p53+/+ cells, but urinary concentrating ability of p53−/− mice is normal, as is the histology of inner medullas from p53−/− and Gadd45abg−/− mice. Thus although Gadd45 and p53 may play roles in osmotically stressed mIMCD3 cells, we do not find that their expression makes an important difference, either for Gadd45 in slower proliferating p2mIME cells or for Gadd45 or p53 in normal inner medullary epithelial cells in situ.

scholarly journals Rapid Evaluation of CRISPR Guides and Donors for Engineering Mice

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 628
Author(s):  
Elena McBeath ◽  
Jan Parker-Thornburg ◽  
Yuka Fujii ◽  
Neeraj Aryal ◽  
Chad Smith ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Sanger Sequencing ◽  
High Efficiency ◽  
Genetically Engineered ◽  
Rapid Evaluation ◽  
Knock Out ◽  
Guide Rna ◽  
Guide Rnas ◽  
Genetically Engineered Mice ◽  
The Cost

Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut.

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