scholarly journals CRISPR-finder: A high throughput and cost-effective method to identify successfully edited Arabidopsis thaliana individuals

2021 ◽  
Vol 2 ◽  
Author(s):  
Efthymia Symeonidi ◽  
Julian Regalado ◽  
Rebecca Schwab ◽  
Detlef Weigel
Keyword(s):  
Arabidopsis Thaliana ◽  
Salicylic Acid ◽  
High Throughput ◽  
Cost Effective ◽  
Repair Process ◽  
Wild Type ◽  
Isochorismate Synthase ◽  
Guide Rna ◽  
Cost Effective Method ◽  
Variable Efficiency

Abstract Genome editing with the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated protein) system allows mutagenesis of a targeted region of the genome using a Cas endonuclease and an artificial guide RNA. Both because of variable efficiency with which such mutations arise and because the repair process produces a spectrum of mutations, one needs to ascertain the genome sequence at the targeted locus for many individuals that have been subjected to mutagenesis. We provide a complete protocol for the generation of amplicons up until the identification of the exact mutations in the targeted region. CRISPR-finder can be used to process thousands of individuals in a single sequencing run. We successfully identified an ISOCHORISMATE SYNTHASE 1 mutant line in which the production of salicylic acid was impaired compared to the wild type, as expected. These features establish CRISPR-finder as a high-throughput, cost-effective and efficient genotyping method of individuals whose genomes have been targeted using the CRISPR/Cas9 system.

scholarly journals CRISPR-finder: A high throughput and cost effective method for identifying successfully edited A. thaliana individuals

2020 ◽  
Author(s):  
Efthymia Symeonidi ◽  
Julian Regalado ◽  
Rebecca Schwab ◽  
Detlef Weigel
Keyword(s):  
High Throughput ◽  
Sanger Sequencing ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Repair Process ◽  
Wild Type ◽  
Isochorismate Synthase ◽  
Guide Rna ◽  
Cost Effective Method ◽  
Variable Efficiency

AbstractBackgroundGenome editing with the CRISPR/Cas9 system allows the user to mutate a targeted region of the genome using an endonuclease (Cas9) and an artificial single-guide RNA (sgRNA). Both because of variable efficiency with which such mutations arise and because the repair process produces a spectrum of mutations, one needs to ascertain the genome sequence at the targeted locus for many individuals that have been subjected to CRISPR/Cas9 mutagenesis. This process can be laborious, expensive and inefficient with conventional methods such as the T7E1 assay or Sanger sequencing. An alternative comprises methods for amplicon sequencing, but most available protocols do not include a facile way for high throughput generation of the samples for sequencing.ResultsIn this study we provide a full pipeline based on amplicon sequencing, CRISPR-finder. We provide a complete protocol for the generation of amplicons up until the identification of the exact mutations in the targeted region. CRISPR-finder can be used to process thousands of individuals in a single sequencing run. For example, we were able to analyze in one sequencing reaction over 900 Arabidopsis thaliana individuals whose genomes had been targeted with the CRISPR/Cas9 system.ConclusionsIn order to validate the potential of CRISPR-finder, we targeted the ISOCHORISMATE SYNTHASE 1 gene in A. thaliana using the CRISPR/Cas9 system. We successfully identified a mutant line in which the production of salicylic acid was impaired compared to the wild type, as expected. These features establish CRISPR-finder as a high-throughput, cost-effective and -efficient genotyping method of individuals whose genomes have been targeted using the CRISPR/Cas9 system.

scholarly journals Simple, Fast, and Cost-Effective Fabrication of Wafer-Scale Nanohole Arrays on Silicon for Antireflection

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Di Di ◽  
Xuezhong Wu ◽  
Peitao Dong ◽  
Chaoguang Wang ◽  
Jian Chen ◽  
...  
Keyword(s):  
High Throughput ◽  
Crystalline Silicon ◽  
Colloidal Crystal ◽  
Cost Effective ◽  
Wafer Scale ◽  
Crystalline Silicon Solar Cells ◽  
Cost Effective Method ◽  
Nanohole Arrays ◽  
Coating Method ◽  
Hole Arrays

A simple, fast, and cost-effective method was developed in this paper for the high-throughput fabrication of nanohole arrays on silicon (Si), which is utilized for antireflection. Wafer-scale polystyrene (PS) monolayer colloidal crystal was developed as templates by spin-coating method. Metallic shadow mask was prepared by lifting off the oxygen etched PS beads from the deposited chromium film. Nanohole arrays were fabricated by Si dry etching. A series of nanohole arrays were fabricated with the similar diameter but with different depth. It is found that the maximum depth of the Si-hole was determined by the diameter of the Cr-mask. The antireflection ability of these Si-hole arrays was investigated. The results show that the reflection decreases with the depth of the Si-hole. The deepest Si-hole arrays show the best antireflection ability (reflection < 9%) at long wavelengths (>600 nm), which was about 28 percent of the nonpatterned silicon wafer’s reflection. The proposed method has the potential for high-throughput fabrication of patterned Si wafer, and the low reflectivity allows the application of these wafers in crystalline silicon solar cells.

scholarly journals Genetic Analysis of colorectal carcinoma using high throughput SNP genotyping technique within the population of Jammu and Kashmir

2021 ◽  
Author(s):  
Bhanu Sharma ◽  
Shabab Angurana ◽  
Amrita Bhat ◽  
Sonali Verma ◽  
Divya Bakshi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
High Throughput ◽  
Cost Effective ◽  
Snp Genotyping ◽  
Colorectal Carcinogenesis ◽  
P Value ◽  
Jammu And Kashmir ◽  
Cost Effective Method ◽  
Underlying Causes ◽  
Multiple Samples

Abstract Background SNP genotyping has become increasingly more common place to understand the genetic basis of complex diseases like cancer. SNP-genotyping through massARRAY is a cost-effective method to quantitatively analyse the variation of gene expression in multiple samples, making it a potential tool to identify the underlying causes of colorectal carcinogenesis. Methods In the present study, SNP genotyping was carried out using Agena mass ARRAY, which is a cost-effective, robust, and sensitive method to analyse multiple SNPs simultaneously. We analysed 7 genes in 492 samples (100 cases and 392 controls) associated with CRC within the population of Jammu and Kashmir. These SNPs were selected based on their association with multiple cancers in literature. Results This is the first study to explore these SNPs with colorectal cancer within the J&K population.7 SNPs with a call rate of 90% were selected for the study. Out of these, one SNP i.e. rs2229080 of DCC was found to be significantly associated with the current study and 6 were non-significantly associated with CRC within the studied population. The allelic OR observed for the variant rs2229080 of DCC was 1.5 (1.1–2.3 at 95% CI), p value = 0.02. Conclusion This is the first study to find the relation of Genetic variants with the colorectal cancer within the studied population using high throughput mass ARRAY technology. It is further anticipated that the variants should be evaluated in other population groups that may aid in understanding the genetic complexity and bridge the missing heritability.

Wire mask assisted rolling as a cost-effective method for high-throughput surface micro-texturing

2020 ◽  
Vol 30 (7) ◽  
pp. 075010
Author(s):  
Anvesh Gaddam ◽  
Ashwin Prabhakaran ◽  
Amit Agrawal ◽  
Suhas S Joshi
Keyword(s):  
High Throughput ◽  
Cost Effective ◽  
Cost Effective Method

scholarly journals Genetic Analysis of colorectal carcinoma using high throughput SNP genotyping technique within the population of Jammu and Kashmir.

2021 ◽  
Author(s):  
Bhanu Sharma ◽  
Shabab Angurana ◽  
Amrita Bhat ◽  
Sonali Verma ◽  
Divya Bakshi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
High Throughput ◽  
Cost Effective ◽  
Snp Genotyping ◽  
Colorectal Carcinogenesis ◽  
P Value ◽  
Jammu And Kashmir ◽  
Cost Effective Method ◽  
Underlying Causes ◽  
Multiple Samples

Abstract Background SNP genotyping has become increasingly more common place to understand the genetic basis of complex diseases like cancer. SNP-genotyping through massARRAY is a cost-effective method to quantitatively analyse the variation of gene expression in multiple samples, making it a potential tool to identify the underlying causes of colorectal carcinogenesis.Methods In the present study, SNP genotyping was carried out using Agena mass ARRAY, which is a cost-effective, robust, and sensitive method to analyse multiple SNPs simultaneously. We analysed 7 genes in 492 samples (100 cases and 392 controls) associated with CRC within the population of Jammu and Kashmir. These SNPs were selected based on their association with multiple cancers in literature.Results This is the first study to explore these SNPs with colorectal cancer within the J&K population.7 SNPs with a call rate of 90% were selected for the study. Out of these, one SNP i.e. rs2229080 of DCC was found to be significantly associated with the current study and 6 were non-significantly associated with CRC within the studied population. The allelic OR observed for the variant rs2229080 of DCC was 1.5 (1.1–2.3 at 95% CI), p value = 0.02.Conclusion This is the first study to find the relation of Genetic variants with the colorectal cancer within the studied population using high throughput mass ARRAY technology. It is further anticipated that the variants should be evaluated in other population groups that may aid in understanding the genetic complexity and bridge the missing heritability.

scholarly journals MultiFRAGing: Rapid and Simultaneous Genotyping of Multiple Alleles in a Single Reaction

2019 ◽  
Author(s):  
Cassidy Petree ◽  
Gaurav K Varshney
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Large Scale ◽  
Cost Effective ◽  
Fold Increase ◽  
Targeted Mutagenesis ◽  
Model Organisms ◽  
Multiple Alleles ◽  
Cost Effective Method ◽  
Single Reaction

AbstractThe powerful and simple RNA-guided CRISPR/Cas9 technology is a versatile genome editing tool that has revolutionized targeted mutagenesis. CRISPR-based genome editing has enabled large-scale functional genetic studies through the generation of gene knockouts in a variety of model organisms including zebrafish. CRISPR/Cas9 can also be used to target multiple genes simultaneously. One of the challenges associated with applying this technique to zebrafish in a high-throughput manner is the absence of a cost-effective method by which to identify mutants. To address this, we optimized the high-throughput, high-resolution fluorescent PCR-based fragment analysis method to develop MultiFRAGing, a robust and cost-effective method for genotyping of multiple targets in a single reaction. Our approach can identify indels in 4 targets from a single reaction, which represents a four-fold increase in genotyping throughput. This method can be used by any laboratory with access to capillary electrophoresis based sequencing equipment.

scholarly journals Knockdown of Quinolinate Phosphoribosyltransferase Results in Decreased Salicylic Acid-Mediated Pathogen Resistance in Arabidopsis thaliana

2021 ◽  
Vol 22 (16) ◽  
pp. 8484
Author(s):  
Shengchun Li ◽  
Haiyan Ding ◽  
Yi Deng ◽  
Jiang Zhang
Keyword(s):  
Arabidopsis Thaliana ◽  
Salicylic Acid ◽  
Pseudomonas Syringae ◽  
De Novo ◽  
Oxidative Stress Marker ◽  
Marker Genes ◽  
Avirulent Strain ◽  
Wild Type ◽  
Nad Biosynthesis ◽  
Quinolinate Phosphoribosyltransferase

Nicotinamide adenine dinucleotide (NAD) is a pivotal coenzyme that has emerged as a central hub linking redox equilibrium and signal transduction in living cells. The homeostasis of NAD is required for plant growth, development, and adaption to environmental stresses. Quinolinate phosphoribosyltransferase (QPRT) is a key enzyme in NAD de novo synthesis pathway. T-DNA-based disruption of QPRT gene is embryo lethal in Arabidopsis thaliana. Therefore, to investigate the function of QPRT in Arabidopsis, we generated transgenic plants with decreased QPRT using the RNA interference approach. While interference of QPRT gene led to an impairment of NAD biosynthesis, the QPRT RNAi plants did not display distinguishable phenotypes under the optimal condition in comparison with wild-type plants. Intriguingly, they exhibited enhanced sensitivity to an avirulent strain of Pseudomonas syringae pv. tomato (Pst-avrRpt2), which was accompanied by a reduction in salicylic acid (SA) accumulation and down-regulation of pathogenesis-related genes expression as compared with the wild type. Moreover, oxidative stress marker genes including GSTU24, OXI1, AOX1 and FER1 were markedly repressed in the QPRT RNAi plants. Taken together, these data emphasized the importance of QPRT in NAD biosynthesis and immunity defense, suggesting that decreased antibacterial immunity through the alteration of NAD status could be attributed to SA- and reactive oxygen species-dependent pathways.

Tissue Macroarray: A Simple and Cost-effective Method for High-Throughput Studies

2003 ◽  
Vol 11 (2) ◽  
pp. 174-176 ◽  
Author(s):  
Liang Wang ◽  
Michael T. Deavers ◽  
Anais Malpica ◽  
Elvio G. Silva ◽  
Jinsong Liu
Keyword(s):  
High Throughput ◽  
Cost Effective ◽  
Cost Effective Method
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