scholarly journals Bar-seq strategies for the LeishGEdit toolbox

2020 ◽  
Author(s):  
Tom Beneke ◽  
Eva Gluenz
Keyword(s):  
High Throughput ◽  
Gene Function ◽  
Gene Editing ◽  
Primer Design ◽  
List Type ◽  
Loss Of Function ◽  
Link Type ◽  
Analysis Tools ◽  
Mixed Populations

AbstractThe number of fully sequenced genomes increases steadily but the function of many genes remains unstudied. To accelerate dissection of gene function in Leishmania spp. and other kinetoplastids we developed previously a streamlined pipeline for CRISPR-Cas9 gene editing, which we termed LeishGEdit [1]. To facilitate high-throughput mutant screens we have adapted this pipeline by barcoding mutants with unique 17-nucleotide barcodes, allowing loss-of-function screens in mixed populations [2]. Here we present primer design and analysis tools that facilitate these bar-seq strategies. We have developed a standalone easy-to-use pipeline to design CRISPR primers suitable for the LeishGEdit toolbox for any given genome and have generated a list of 14,995 barcodes. Barcodes and oligos are now accessible through our website www.leishgedit.net allowing to pursue bar-seq experiments in all currently available TriTrypDB genomes (release 41). This will streamline CRISPR bar-seq assays in kinetoplastids, enabling pooled mutant screens across the community.HighlightsDeveloping tools for pooled bar-seq mutant screens across the kinetoplastid communityDevelopment of a standalone script to design primers suitable for the LeishGEdit toolboxGeneration of 14,995 barcodes that can be used for bar-seq strategies in kinetoplastidsBar-seq primers for all TriTrypDB genomes (release 41) can be obtained from www.leishgedit.net

scholarly journals Phenotype prediction in anEscherichia colistrain panel

2017 ◽  
Author(s):  
Marco Galardin ◽  
Alexandra Koumoutsi ◽  
Lucia Herrera-Dominguez ◽  
Juan Antonio Cordero Varela ◽  
Anja Telzerow ◽  
...  
Keyword(s):  
High Throughput ◽  
Gene Function ◽  
Genetic Variants ◽  
List Type ◽  
Loss Of Function ◽  
Phenotypic Differences ◽  
E Coli ◽  
Growth Defects ◽  
K 12 ◽  
The Impact

SummaryUnderstanding how genetic variation contributes to phenotypic differences is a fundamental question in biology. Here, we set to predict fitness defects of an individual using mechanistic models of the impact of genetic variants combined with prior knowledge of gene function. We assembled a diverse panel of 696Escherichia colistrains for which we obtained genomes and measured growth phenotypes in 214 conditions. We integrated variant effect predictors to derive gene-level probabilities of loss of function for every gene across strains. We combined these probabilities with information on conditional gene essentiality in the reference K-12 strain to predict the strains’ growth defects, providing significant predictions for up to 38% of tested conditions. The putative causal variants were validated in complementation assays highlighting commonly perturbed pathways in evolution for the emergence of growth phenotypes. Altogether, our work illustrates the power of integrating high-throughput gene function assays to predict the phenotypes of individuals.HighlightsAssembled a reference panel ofE. colistrainsGenotyped and high-throughput phenotyped theE. colireference strain panelReliably predicted the impact of genetic variants in up to 38% of tested conditionsHighlighted common genetic pathways for the emergence of deleterious phenotypes

scholarly journals Proxies of CRISPR/Cas9 Activity To Aid in the Identification of Mutagenized Arabidopsis Plants

2020 ◽  
Vol 10 (6) ◽  
pp. 2033-2042 ◽  
Author(s):  
Renyu Li ◽  
Charles Vavrik ◽  
Cristian H. Danna
Keyword(s):  
Arabidopsis Thaliana ◽  
Gene Function ◽  
Good Predictor ◽  
Gene Editing ◽  
Selection Strategy ◽  
Directed Mutagenesis ◽  
Loss Of Function ◽  
Editing Event ◽  
Endogenous Gene ◽  
Non Coding Rnas

CRISPR/Cas9 has become the preferred gene-editing technology to obtain loss-of-function mutants in plants, and hence a valuable tool to study gene function. This is mainly due to the easy reprogramming of Cas9 specificity using customizable small non-coding RNAs, and to the possibility of editing several independent genes simultaneously. Despite these advances, the identification of CRISPR-edited plants remains time and resource-intensive. Here, based on the premise that one editing event in one locus is a good predictor of editing event/s in other locus/loci, we developed a CRISPR co-editing selection strategy that greatly facilitates the identification of CRISPR-mutagenized Arabidopsis thaliana plants. This strategy is based on targeting the gene/s of interest simultaneously with a proxy of CRISPR-Cas9-directed mutagenesis. The proxy is an endogenous gene whose loss-of-function produces an easy-to-detect visible phenotype that is unrelated to the expected phenotype of the gene/s under study. We tested this strategy via assessing the frequency of co-editing of three functionally unrelated proxy genes. We found that each proxy predicted the occurrence of mutations in each surrogate gene with efficiencies ranging from 68 to 100%. The selection strategy laid out here provides a framework to facilitate the identification of multiplex edited plants, thus aiding in the study of gene function when functional redundancy hinders the effort to define gene-function-phenotype links.

scholarly journals AmpliconDesign – An interactive web server for the design of high-throughput targeted DNA methylation assays

2020 ◽  
Author(s):  
Maximilian Schönung ◽  
Jana Hess ◽  
Pascal Bawidamann ◽  
Sina Stäble ◽  
Joschka Hey ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Quality Control ◽  
High Throughput ◽  
Dna Sequences ◽  
Web Server ◽  
Dna Amplification ◽  
Pcr Primers ◽  
Primer Design ◽  
Link Type ◽  
Targeted Analysis

ABSTRACTTargeted analysis of DNA methylation patterns based on bisulfite-treated genomic DNA (BT-DNA) is considered as a gold-standard for epigenetic biomarker development. Existing software tools facilitate primer design, primer quality control or visualization of primer localization. However, high-throughput design of primers for BT-DNA amplification is hampered by limits in throughput and functionality of existing tools, requiring users to repeatedly perform specific tasks manually. Consequently, the design of PCR primers for BT-DNA remains a tedious and time-consuming process. To bridge this gap, we developed AmpliconDesign, a webserver providing a scalable and user-friendly platform for the design and analysis of targeted DNA methylation studies based on BT-DNA, e.g. deep amplicon bisulfite sequencing (ampBS-seq), EpiTYPER MassArray, or pyrosequencing. Core functionality of the web server includes high-throughput primer design and binding site validation based on in silico bisulfite-converted DNA sequences, prediction of fragmentation patterns for EpiTYPER MassArray, an interactive quality control as well as a streamlined analysis workflow for ampBS-seq.Availability and ImplementationThe AmpliconDesign webserver is freely available online at: https://amplicondesign.dkfz.de/. AmpliconDesign has been implemented using the R Shiny framework (Chang et al., 2018). The source code is publicly available under the GNU General Public License v3.0 (https://github.com/MaxSchoenung/AmpliconDesign).ContactDaniel B. Lipka ([email protected]) & Maximilian Schönung ([email protected])

scholarly journals CRISPR/Cas9‐Directed Gene Editing for the Generation of Loss‐of‐Function Mutants in High‐Throughput Zebrafish F 0 Screens

2017 ◽  
Vol 119 (1) ◽  
Author(s):  
Sunita S. Shankaran ◽  
Timothy J. Dahlem ◽  
Brent W. Bisgrove ◽  
H. Joseph Yost ◽  
Martin Tristani‐Firouzi
Keyword(s):  
High Throughput ◽  
Gene Editing ◽  
Loss Of Function

scholarly journals PrimerServer: a high-throughput primer design and specificity-checking platform

2017 ◽  
Author(s):  
Tao Zhu ◽  
Chengzhen Liang ◽  
Zhigang Meng ◽  
Yanyan Li ◽  
Yayu Wu ◽  
...  
Keyword(s):  
High Throughput ◽  
Molecular Breeding ◽  
Primer Design ◽  
Command Line ◽  
Specific Primers ◽  
Data Presentation ◽  
Medical Testing ◽  
Link Type ◽  
Fast Running

AbstractSummaryDesigning specific primers for multiple sites across the whole genome is still challenging, especially in species with complex genomes. Here we present PrimerServer, a high-throughput primer design and specificity-checking platform with both web and command-line interfaces. This platform efficiently integrates site selection, primer design, specificity checking and data presentation. In our case study, PrimerServer achieved high accuracy and a fast running speed for a large number of sites, suggesting its potential for molecular biology applications such as molecular breeding or medical testing.Availability and ImplementationSource code for PrimerServer is available at https://github.com/billzt/PrimerServer. A demo server is freely accessible at https://primerserver.org, with all major browsers [email protected] or [email protected]

scholarly journals Robust high throughput prokaryote de novo assembly and improvement pipeline for Illumina data

2016 ◽  
Author(s):  
Andrew J. Page ◽  
Nishadi De Silva ◽  
Martin Hunt ◽  
Michael A. Quail ◽  
Julian Parkhill ◽  
...  
Keyword(s):  
Open Source ◽  
High Throughput ◽  
De Novo ◽  
Bacterial Genome ◽  
List Type ◽  
Genome Diversity ◽  
Link Type ◽  
Open Source License ◽  
Public Data ◽  
Genome Assemblies

ABSTRACTThe rapidly reducing cost of bacterial genome sequencing has lead to its routine use in large scale microbial analysis. Though mapping approaches can be used to find differences relative to the reference, many bacteria are subject to constant evolutionary pressures resulting in events such as the loss and gain of mobile genetic elements, horizontal gene transfer through recombination and genomic rearrangements. De novo assembly is the reconstruction of the underlying genome sequence, an essential step to understanding bacterial genome diversity. Here we present a high throughput bacterial assembly and improvement pipeline that has been used to generate nearly 20,000 draft genome assemblies in public databases. We demonstrate its performance on a public data set of 9,404 genomes. We find all the genes used in MLST schema present in 99.6% of assembled genomes. When tested on low, neutral and high GC organisms, more than 94% of genes were present and completely intact. The pipeline has proven to be scalable and robust with a wide variety of datasets without requiring human intervention. All of the software is available on GitHub under the GNU GPL open source license.DATA SUMMARYThe assembly pipeline software is available from Github under the GNU GPL open source license; (url - https://github.com/sanger-pathogens/vr-codebase)The assembly improvement software is available from Github under the GNU GPL open source license; (url - https://github.com/sanger-pathogens/assembly_improvement)Accession numbers for 9,404 assemblies are provided in the supplementary material.The Bordetella pertussis sample has sample accession ERS1058649, sequencing reads accession ERR1274624 and assembly accessions FJMX01000001-FJMX01000249.The Salmonella enterica subsp. enterica serovar Pullorum sample has sample accession ERS1058652, sequencing reads accession ERR1274625 and assembly accession FJMV01000001-FJMV01000026.The Staphylococcus aureus sample has sample accession ERS1058648, sequencing reads accession ERR1274626 and assembly accessions FJMW01000001-FJMW01000040.I/We confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.☑IMPACT STATEMENTThe pipeline described in this paper has been used to assemble and annotate 30% of all bacterial genome assemblies in GenBank (18,080 out of 59,536, accessed 16/2/16). The automated generation of de novo assemblies is a critical step to explore bacterial genome diversity. MLST genes are found in 99.6% of cases, making it at least as good as existing typing methods. In the test genomes we present, more than 94% of genes are correctly assembled into intact reading frames.

scholarly journals High throughput single-cell detection of multiplex CRISPR-edited gene modifications

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Elisa ten Hacken ◽  
Kendell Clement ◽  
Shuqiang Li ◽  
María Hernández-Sánchez ◽  
Robert Redd ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Single Cell ◽  
High Throughput ◽  
Gene Editing ◽  
Somatic Mutations ◽  
Single Cells ◽  
Lymphocytic Leukemia ◽  
Cell Detection ◽  
Loss Of Function ◽  
Single Cell Detection

Abstract CRISPR-Cas9 gene editing has transformed our ability to rapidly interrogate the functional impact of somatic mutations in human cancers. Droplet-based technology enables the analysis of Cas9-introduced gene edits in thousands of single cells. Using this technology, we analyze Ba/F3 cells engineered to express single or multiplexed loss-of-function mutations recurrent in chronic lymphocytic leukemia. Our approach reliably quantifies mutational co-occurrences, zygosity status, and the occurrence of Cas9 edits at single-cell resolution.

scholarly journals Proxies of CRISPR-Cas9 activity to aid in the identification of mutagenized Arabidopsis plants

2019 ◽  
Author(s):  
Renyu Li ◽  
Charles Vavrik ◽  
Cristian H. Danna
Keyword(s):  
Gene Function ◽  
Gene Editing ◽  
Gene Families ◽  
Single Copy ◽  
Selection Strategy ◽  
Loss Of Function ◽  
Editing Event ◽  
Endogenous Gene ◽  
Polyploid Plant ◽  
Non Coding Rnas

AbstractCRISPR-Cas9 has become the preferred gene editing technology to obtain loss-of-function mutants in plants, and hence a valuable tool to study gene function. This is mainly due to the easy reprograming of Cas9 specificity using customizable small non-coding RNAs, and to the ability to target several independent genes simultaneously. Despite these advances, the identification of CRISPR-edited plants remains time and resource consuming. Here, based on the premise that one editing event in one locus is a good predictor of editing event/s in other locus/loci, we developed a CRISPR co-editing selection strategy that greatly facilitates the identification of CRISPR-mutagenized Arabidopsis plants. This strategy is based on targeting the gene/s of interest simultaneously with a proxy of CRISPR-Cas9-directed mutagenesis. The proxy is an endogenous gene whose loss-of-function mutation produces an easy-to-detect visible phenotype that is unrelated to the expected phenotype of the gene/s under study. We tested this strategy via assessing the frequency of co-editing of three functionally unrelated proxies. We found all three proxies predicted the occurrence of mutations in either or both of the other two proxies with efficiencies ranging from 40% to 100%, dramatically reducing the number of plants that need to be screened to identify CRISPR mutants. This selection strategy provides a framework to facilitate gene function studies of gene families as well as the function of single copy genes in polyploid plant species where the identification of multiplex mutants remains challenging.

scholarly journals The NALCN Channel Regulator UNC-80 Functions in a Subset of Interneurons To Regulate Caenorhabditis elegans Reversal Behavior

2019 ◽  
Vol 10 (1) ◽  
pp. 199-210 ◽  
Author(s):  
Chuanman Zhou ◽  
Jintao Luo ◽  
Xiaohui He ◽  
Qian Zhou ◽  
Yunxia He ◽  
...  
Keyword(s):  
Plant Hormone ◽  
Neuronal Excitability ◽  
Resting Membrane Potential ◽  
Loss Of Function ◽  
Wild Type ◽  
C Elegans ◽  
Link Type ◽  
Complex Functions ◽  
And Function ◽  
Multiple Loss

NALCN (Na+leak channel, non-selective) is a conserved, voltage-insensitive cation channel that regulates resting membrane potential and neuronal excitability. UNC79 and UNC80 are key regulators of the channel function. However, the behavioral effects of the channel complex are not entirely clear and the neurons in which the channel functions remain to be identified. In a forward genetic screen for C. elegans mutants with defective avoidance response to the plant hormone methyl salicylate (MeSa), we isolated multiple loss-of-function mutations in unc-80 and unc-79. C. elegans NALCN mutants exhibited similarly defective MeSa avoidance. Interestingly, NALCN, unc-80 and unc-79 mutants all showed wild type-like responses to other attractive or repelling odorants, suggesting that NALCN does not broadly affect odor detection or related forward and reversal behaviors. To understand in which neurons the channel functions, we determined the identities of a subset of unc-80-expressing neurons. We found that unc-79 and unc-80 are expressed and function in overlapping neurons, which verified previous assumptions. Neuron-specific transgene rescue and knockdown experiments suggest that the command interneurons AVA and AVE and the anterior guidepost neuron AVG can play a sufficient role in mediating unc-80 regulation of the MeSa avoidance. Though primarily based on genetic analyses, our results further imply that MeSa might activate NALCN by direct or indirect actions. Altogether, we provide an initial look into the key neurons in which the NALCN channel complex functions and identify a novel function of the channel in regulating C. elegans reversal behavior through command interneurons.

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