scholarly journals PrimeDesign software for rapid and simplified design of prime editing guide RNAs

2020 ◽  
Author(s):  
Jonathan Hsu ◽  
Julian Grünewald ◽  
Regan Szalay ◽  
Justine Shih ◽  
Andrew Anzalone ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Web Application ◽  
Saturation Mutagenesis ◽  
Command Line ◽  
Guide Rna ◽  
Guide Rnas ◽  
Pathogenic Variants ◽  
Genome Wide ◽  
Command Line Tool ◽  
User Friendly

Abstract Prime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we constructed PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we used PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells.

scholarly journals PrimeDesign software for rapid and simplified design of prime editing guide RNAs

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jonathan Y. Hsu ◽  
Julian Grünewald ◽  
Regan Szalay ◽  
Justine Shih ◽  
Andrew V. Anzalone ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Web Application ◽  
Saturation Mutagenesis ◽  
Command Line ◽  
Guide Rna ◽  
Guide Rnas ◽  
Pathogenic Variants ◽  
Genome Wide ◽  
Command Line Tool ◽  
User Friendly

AbstractPrime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we construct PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we use PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells.

scholarly journals PrimeDesign software for rapid and simplified design of prime editing guide RNAs

2020 ◽  
Author(s):  
Jonathan Y. Hsu ◽  
Andrew V. Anzalone ◽  
Julian Grünewald ◽  
Kin Chung Lam ◽  
Max W. Shen ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Web Application ◽  
Saturation Mutagenesis ◽  
Command Line ◽  
Searchable Database ◽  
Guide Rna ◽  
Guide Rnas ◽  
Genome Wide ◽  
Command Line Tool ◽  
User Friendly

AbstractPrime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we also constructed PrimeVar, the first comprehensive and searchable database for prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations to install or correct >68,500 pathogenic human genetic variants from the ClinVar database.

scholarly journals GuideMaker: Software to design CRISPR-Cas guide RNA pools in non-model genomes

2021 ◽  
Author(s):  
Ravin Poudel ◽  
Lidimarie Trujillo Rodriguez ◽  
Christopher R Reisch ◽  
Adam R Rivers
Keyword(s):  
Web Application ◽  
Bacterial Genome ◽  
Small World ◽  
Design Tool ◽  
Model Organisms ◽  
Guide Rna ◽  
Creative Commons ◽  
Guide Rnas ◽  
Genome Wide ◽  
Protospacer Adjacent Motif

Background: CRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing the CRISPR-Cas guide RNAs for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for atypical projects with 1) non-standard Cas enzymes, 2) non-model organisms, or 3) projects that need to design a panel of guide RNAs (gRNA) for genome-wide screens. Findings: GuideMaker can rapidly design gRNAs for gene targets across the genome from a degenerate protospacer adjacent motif (PAM) and a GenBank file. The tool applies Hierarchical Navigable Small World (HNSW) graphs to speed up the comparison of guide RNAs. This allows the user to design gRNAs targeting all genes in a typical bacterial genome in about 1-2 minutes. Conclusions: Guidemaker enables the rapid design of genome-wide gRNA for any CRISPR-Cas enzyme in non-model organisms. While GuideMaker is designed with prokaryotic genomes in mind, it can efficiently process smaller eukaryotic genomes as well. GuideMaker is available as command-line software, a stand-alone web application, and a tool in the CyCverse Discovery Environment. All versions are available under a Creative Commons CC0 1.0 Universal Public Domain Dedication.

scholarly journals pegFinder: A pegRNA designer for CRISPR prime editing

2020 ◽  
Author(s):  
Ryan D. Chow ◽  
Jennifer S. Chen ◽  
Johanna Shen ◽  
Sidi Chen
Keyword(s):  
Web Application ◽  
Double Strand Breaks ◽  
Primer Binding Site ◽  
Web Tool ◽  
Strand Breaks ◽  
Guide Rna ◽  
Guide Rnas ◽  
Target Dna ◽  
Target Sites ◽  
User Friendly

To the EditorCRISPR technologies have been widely adopted as powerful tools for targeted genomic manipulation 1. Recently, a new CRISPR-based strategy for precision genome editing was developed that enables diverse genomic alterations to be directly written into target sites without requiring double-strand breaks (DSBs) or donor templates 2. Termed prime editing, this approach involves two key components: 1) a catalytically impaired Cas9 nickase fused to a reverse transcriptase (PE2), and 2) a multifunctional prime editing guide RNA (pegRNA) that specifies the target site and further acts as a template for reverse transcription (RT). pegRNAs are similar to standard single-guide RNAs (sgRNAs), but additionally have a customizable extension on the 3’ end. The 3’ extension is composed of a RT template that encodes the desired edit and a primer binding site (PBS) that anneals to the target genomic site to prime the RT reaction 2. These additional components considerably increase the complexity of pegRNA design compared to standard sgRNAs. While many tools have been developed for identifying candidate sgRNAs in a target DNA sequence 3–8, no user-friendly web application currently exists for designing pegRNAs. We therefore developed pegFinder, a streamlined web tool that rapidly designs candidate pegRNAs (Figure 1). The pegFinder web portal is freely available at http://pegfinder.sidichenlab.org/ (Supplementary Figure 1).

scholarly journals Gene-based association analysis identified 190 genes with polymorphisms affecting neuroticism

2020 ◽  
Author(s):  
Nadezhda M. Belonogova ◽  
Irina V. Zorkoltseva ◽  
Yakov A. Tsepilov ◽  
Tatiana I. Axenovich
Keyword(s):  
Gene Expression ◽  
Detailed Analysis ◽  
Association Analysis ◽  
Genetic Variants ◽  
Bioinformatics Analysis ◽  
Summary Statistics ◽  
Uk Biobank ◽  
Protein Coding ◽  
Pathogenic Variants ◽  
Genome Wide

AbstractRecent genome-wide studies have reported about 600 genes potentially influencing neuroticism. Little is known about the mechanisms of their action. Here, we aimed to conduct a more detailed analysis of genes whose polymorphisms can regulate the level of neuroticism. Using UK Biobank-based GWAS summary statistics, we performed a gene-based association analysis using four sets of genetic variants within a gene differing in their protein coding properties. To guard against the influence of strong GWAS signals outside the gene, we used the specially designed procedure. As a result, we identified 190 genes associated with neuroticism due to their polymorphisms. Thirty eight of these genes were novel. Within all genes identified, we distinguished two slightly overlapping groups comprising genes that demonstrated association when using protein-coding and non-coding SNPs. Many genes from the first group included potentially pathogenic variants. For some genes from the second group, we found evidence of pleiotropy with gene expression. We demonstrated that the association of almost two hundred known genes could be inflated by the GWAS signals outside the gene. Using bioinformatics analysis, we prioritized the neuroticism genes and showed that the genes influencing the trait by their polymorphisms are the most appropriate candidate genes.

scholarly journals Ares-GT: design of guide RNAs targeting multiple genes for CRISPR-Cas experiments

2020 ◽  
Author(s):  
Eugenio G. Minguet
Keyword(s):  
Reference Genome ◽  
Query Sequence ◽  
Gene Families ◽  
Guide Rna ◽  
Guide Rnas ◽  
Online Tools ◽  
Multiple Input ◽  
Design Guide ◽  
Command Line Tool ◽  
Selection Of

ABSTRACTMotivationThere is a lack of tools to design guide RNA for CRISPR genome editing of gene families and usually good candidate sgRNAs are tagged with low scores precisely because they match several locations in the genome, thus time-consuming manual evaluation of targets is required. Moreover, online tools are limited to a restricted list of reference genome and lack the flexibility to incorporate unpublished genomes or contemplate genomes of populations with allelic variants.ResultsTo address these issues, I have developed the ARES-GT, a local command line tool in Python software. ARES-GT allows the selection of candidate sgRNAs that match multiple input query sequences, in addition of candidate sgRNAs that specifically match each query sequence. It also contemplates the use of unmapped contigs apart from complete genomes thus allowing the use of any genome provided by user and being able to handle intraspecies allelic variability and individual polymorphisms.AvailabilityARES-GT is available at GitHub (https://github.com/eugomin/ARES-GT.git).

scholarly journals CRISpy-pop: a web tool for designing CRISPR/Cas9-driven genetic modifications in diverse populations

2020 ◽  
Author(s):  
Hayley R. Stoneman ◽  
Russell L. Wrobel ◽  
Michael Place ◽  
Michael Graham ◽  
David J. Krause ◽  
...  
Keyword(s):  
Web Application ◽  
Genomic Data ◽  
Model Organisms ◽  
Bacterial Strains ◽  
Rna Sequences ◽  
Guide Rna ◽  
Guide Rnas ◽  
Population Genomic ◽  
Current Implementation ◽  
Multiple Strains

AbstractCRISPR/Cas9 is a powerful tool for editing genomes, but design decisions are generally made with respect to a single reference genome. With population genomic data becoming available for an increasing number of model organisms, researchers are interested in manipulating multiple strains and lines. CRISpy-pop is a web application that generates and filters guide RNA sequences for CRISPR/Cas9 genome editing for diverse yeast and bacterial strains. The current implementation designs and predicts the activity of guide RNAs against more than 1000 Saccharomyces cerevisiae genomes, including 167 strains frequently used in bioenergy research. Zymomonas mobilis, an increasingly popular bacterial bioenergy research model, is also supported. CRISpy-pop is available as a web application (https://CRISpy-pop.glbrc.org/) with an intuitive graphical user interface. CRISpy-pop also cross-references the human genome to allow users to avoid the selection of sgRNAs with potential biosafety concerns. Additionally, CRISpy-pop predicts the strain coverage of each guide RNA within the supported strain sets, which aids in functional population genetic studies. Finally, we validate how CRISpy-pop can accurately predict the activity of guide RNAs across strains using population genomic data.

scholarly journals LAVA: a streamlined visualization tool for longitudinal analysis of viral alleles

2019 ◽  
Author(s):  
Michelle J. Lin ◽  
Ryan C. Shean ◽  
Negar Makhsous ◽  
Alexander L. Greninger
Keyword(s):  
Genome Evolution ◽  
Longitudinal Analysis ◽  
Viral Evolution ◽  
Read Depth ◽  
Visualization Tool ◽  
Command Line ◽  
Interactive Analysis ◽  
Command Line Tool ◽  
User Friendly ◽  
Over Time

AbstractWith their small genomes, fast evolutionary rates, and clinical significance, viruses have long been fodder for studies of whole genome evolution. One common need in these studies is the analysis of viral evolution over time through longitudinal sampling. However, there exists no simple tool to automate such analyses. We created a simple command-line visualization tool called LAVA (Longitudinal Analysis of Viral Alleles). LAVA allows dynamic and interactive visualization of viral evolution across the genome and over time. Results are easily shared via a single HTML file that also allows interactive analysis based on read depth and allele frequency. LAVA requires minimal input and runs in minutes for most use cases. LAVA is programmed mainly in Python 3 and is compatible with Mac and Linux machines. LAVA is a user-friendly command-line tool for generating, visualizing, and sharing the results of longitudinal viral genome evolution analysis. Instructions for downloading, installing, and using LAVA can be found at https://github.com/michellejlin/lava.

VEGAS2: Software for More Flexible Gene-Based Testing

2014 ◽  
Vol 18 (1) ◽  
pp. 86-91 ◽  
Author(s):  
Aniket Mishra ◽  
Stuart Macgregor
Keyword(s):  
Association Studies ◽  
Genome Wide Association Studies ◽  
Command Line ◽  
Single Nucleotide ◽  
Web Based ◽  
Reference Set ◽  
Genome Wide ◽  
User Friendly ◽  
Further Development ◽  
Command Line Version

Gene-based tests such as versatile gene-based association study (VEGAS) are commonly used following per-single nucleotide polymorphism (SNP) GWAS (genome-wide association studies) analysis. Two limitations of VEGAS were that the HapMap2 reference set was used to model the correlation between SNPs and only autosomal genes were considered. HapMap2 has now been superseded by the 1,000 Genomes reference set, and whereas early GWASs frequently ignored the X chromosome, it is now commonly included. Here we have developed VEGAS2, an extension that uses 1,000 Genomes data to model SNP correlations across the autosomes and chromosome X. VEGAS2 allows greater flexibility when defining gene boundaries. VEGAS2 offers both a user-friendly, web-based front end and a command line Linux version. The online version of VEGAS2 can be accessed through https://vegas2.qimrberghofer.edu.au/. The command line version can be downloaded from https://vegas2.qimrberghofer.edu.au/zVEGAS2offline.tgz. The command line version is developed in Perl, R and shell scripting languages; source code is available for further development.

scholarly journals CRISpy-Pop: A Web Tool for Designing CRISPR/Cas9-Driven Genetic Modifications in Diverse Populations

2020 ◽  
Vol 10 (11) ◽  
pp. 4287-4294
Author(s):  
Hayley R. Stoneman ◽  
Russell L. Wrobel ◽  
Michael Place ◽  
Michael Graham ◽  
David J. Krause ◽  
...  
Keyword(s):  
Web Application ◽  
Genomic Data ◽  
Model Organisms ◽  
Bacterial Strains ◽  
Rna Sequences ◽  
Guide Rna ◽  
Guide Rnas ◽  
Population Genomic ◽  
Current Implementation ◽  
Multiple Strains

CRISPR/Cas9 is a powerful tool for editing genomes, but design decisions are generally made with respect to a single reference genome. With population genomic data becoming available for an increasing number of model organisms, researchers are interested in manipulating multiple strains and lines. CRISpy-pop is a web application that generates and filters guide RNA sequences for CRISPR/Cas9 genome editing for diverse yeast and bacterial strains. The current implementation designs and predicts the activity of guide RNAs against more than 1000 Saccharomyces cerevisiae genomes, including 167 strains frequently used in bioenergy research. Zymomonas mobilis, an increasingly popular bacterial bioenergy research model, is also supported. CRISpy-pop is available as a web application (https://CRISpy-pop.glbrc.org/) with an intuitive graphical user interface. CRISpy-pop also cross-references the human genome to allow users to avoid the selection of guide RNAs with potential biosafety concerns. Additionally, CRISpy-pop predicts the strain coverage of each guide RNA within the supported strain sets, which aids in functional population genetic studies. Finally, we validate how CRISpy-pop can accurately predict the activity of guide RNAs across strains using population genomic data.

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