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.

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

scholarly journals On-target activity predictions enable improved CRISPR–dCas9 screens in bacteria

2020 ◽  
Vol 48 (11) ◽  
pp. e64-e64 ◽  
Author(s):  
Alicia Calvo-Villamañán ◽  
Jérome Wong Ng ◽  
Rémi Planel ◽  
Hervé Ménager ◽  
Arthur Chen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Cost Effective ◽  
Target Sequence ◽  
Guide Rna ◽  
E Coli ◽  
Genome Wide ◽  
High Throughput Screens ◽  
Target Activity ◽  
Target Effects

Abstract The ability to block gene expression in bacteria with the catalytically inactive mutant of Cas9, known as dCas9, is quickly becoming a standard methodology to probe gene function, perform high-throughput screens, and engineer cells for desired purposes. Yet, we still lack a good understanding of the design rules that determine on-target activity for dCas9. Taking advantage of high-throughput screening data, we fit a model to predict the ability of dCas9 to block the RNA polymerase based on the target sequence, and validate its performance on independently generated datasets. We further design a novel genome wide guide RNA library for E. coli MG1655, EcoWG1, using our model to choose guides with high activity while avoiding guides which might be toxic or have off-target effects. A screen performed using the EcoWG1 library during growth in rich medium improved upon previously published screens, demonstrating that very good performances can be attained using only a small number of well designed guides. Being able to design effective, smaller libraries will help make CRISPRi screens even easier to perform and more cost-effective. Our model and materials are available to the community through crispr.pasteur.fr and Addgene.

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