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 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.

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 Transcriptome-wide Cas13 guide RNA design for model organisms and viral RNA pathogens

2020 ◽  
Author(s):  
Xinyi Guo ◽  
Hans-Hermann Wessels ◽  
Alejandro Méndez-Mancilla ◽  
Daniel Haro ◽  
Neville E. Sanjana
Keyword(s):  
Rna Virus ◽  
H1n1 Influenza ◽  
Model Organisms ◽  
Messenger Rnas ◽  
Protein Coding ◽  
Guide Rna ◽  
Knock Down ◽  
Guide Rnas ◽  
Rna Targeting ◽  
Rna Design

AbstractCRISPR-Cas13 mediates robust transcript knockdown in human cells through direct RNA targeting. Compared to DNA-targeting CRISPR enzymes like Cas9, RNA targeting by Cas13 is transcript- and strand-specific: It can distinguish and specifically knock-down processed transcripts, alternatively spliced isoforms and overlapping genes, all of which frequently serve different functions. Previously, we identified optimal design rules for RfxCas13d guide RNAs (gRNAs), and developed a computational model to predict gRNA efficacy for all human protein-coding genes. However, there is a growing interest to target other types of transcripts, such as noncoding RNAs (ncRNAs) or viral RNAs, and to target transcripts in other commonly-used organisms. Here, we predicted relative Cas13-driven knock-down for gRNAs targeting messenger RNAs and ncRNAs in six model organisms (human, mouse, zebrafish, fly, nematode and flowering plants) and four abundant RNA virus families (SARS-CoV-2, HIV-1, H1N1 influenza and MERS). To allow for more flexible gRNA efficacy prediction, we also developed a web-based application to predict optimal gRNAs for any RNA target entered by the user. Given the lack of Cas13 guide design tools, we anticipate this resource will facilitate CRISPR-Cas13 RNA targeting in common model organisms, emerging viral threats to human health, and novel RNA targets.

scholarly journals dbGuide: A database of functionally validated guide RNAs for genome editing in human and mouse cells

2020 ◽  
Author(s):  
Alexander A. Gooden ◽  
Christine N. Evans ◽  
Timothy P. Sheets ◽  
Michelle E. Clapp ◽  
Raj Chari
Keyword(s):  
Epigenetic Modification ◽  
Gene Activation ◽  
Amplicon Sequencing ◽  
Ease Of Use ◽  
Rna Sequences ◽  
Additional Species ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Human And Mouse

ABSTRACTWith the technology’s accessibility and ease of use, CRISPR has been employed widely in many different organisms and experimental settings. As a result, thousands of publications have used CRISPR to make specific genetic perturbations, establishing in itself a resource of validated guide RNA sequences. While numerous computational tools to assist in the design and identification of candidate guide RNAs exist, these are still just at best predictions and generally, researchers inevitably will test multiple sequences for functional activity. Here, we present dbGuide (https://sgrnascorer.cancer.gov/dbguide), a database of functionally validated guide RNA sequences for CRISPR/Cas9-based knockout in human and mouse. Our database not only contains computationally determined candidate guide RNA sequences, but of even greater value, over 4000 sequences which have been functionally validated either through direct amplicon sequencing or manual curation of literature from over 1000 publications. Finally, our established framework will allow for continual addition of newly published and experimentally validated guide RNA sequences for CRISPR/Cas9-based knockout as well as incorporation of sequences from different gene editing systems, additional species, and other types of site-specific functionalities such as base editing, gene activation, repression, and epigenetic modification.

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 GRIBCG: A software for selection of sgRNAs in the design of balancer chromosomes

2018 ◽  
Author(s):  
Brian B. Merritt ◽  
Lily S. Cheung
Keyword(s):  
Rational Design ◽  
Chromosomal Rearrangements ◽  
Fruit Fly ◽  
Model Organisms ◽  
Guide Rna ◽  
Guide Rnas ◽  
Genetics Research ◽  
Multicellular Organisms ◽  
Balancer Chromosomes ◽  
Selection Of

AbstractBackgroundBalancer chromosomes are tools used by fruit fly geneticists to prevent meiotic recombination. Recently, CRISPR/Cas9 genome editing has been shown capable of generating inversions similar to the chromosomal rearrangements present in balancer chromosomes. Extending the benefits of balancer chromosomes to other multicellular organisms could significantly accelerate biomedical and plant genetics research.ResultsHere, we present GRIBCG (Guide RNA Identifier for Balancer Chromosome Generation), a tool for the rational design of balancer chromosomes. GRIBCG identifies single guide RNAs (sgRNAs) for use with Streptococcus pyogenes Cas9 (SpCas9). These sgRNAs would efficiently cut a chromosome multiple times while minimizing off-target cutting in the rest of the genome. We describe the performance of this tool on six model organisms and compare our results to two routinely used fruit fly balancer chromosomes.ConclusionGRIBCG is the first of its kind tool for the design of balancer chromosomes using CRISPR/Cas9. GRIBCG can accelerate genetics research by providing a fast, systematic and simple to use framework to induce chromosomal rearrangements.

One-step dual CRISPR/Cas9 guide RNA cloning protocol

2019 ◽  
Author(s):  
Anthony Beucher ◽  
Ines Cebola
Keyword(s):  
Regulatory Elements ◽  
Single Step ◽  
Rna Sequences ◽  
Guide Rna ◽  
Guide Rnas ◽  
Dna Oligonucleotides ◽  
Simple Protocol ◽  
Chromatin Structural ◽  
One Step

Abstract Existing protocols for dual guide RNA cloning rely on synthesised DNA oligonucleotides of >100 bp that contain both guide RNA sequences, and are therefore not reusable in alternative experimental designs. Here, we describe a single-step protocol to rapidly and inexpensively generate vectors expressing two guide RNAs (gRNAs) simultaneously, which allows re-usage of gRNAs oligonucleotides from one experimental design to another. This protocol is applicable to cloning gRNAs into virtually any CRISPR/Cas9 backbone that allows cloning by Golden Gate, by adapting the primer design. Here, we provide details for cloning gRNAs into vectors with BbsI and BsmBI sites, two of the most frequently found enzymes in CRISPR/Cas9 gRNA expression cassettes.This protocol has been successfully applied to delete pancreatic islet enhancers that harbour type 2 diabetes variants and to validate enhancer-promoter interactions (Miguel-Escalada et al., Nature Genetics 2019).In the future, we foresee that this simple protocol may also be applied to target coding sequences, as well as to target other important kinds of noncoding regulatory elements, including lncRNAs, miRNAs, and chromatin structural anchor points.

scholarly journals A Guide RNA Sequence Design Platform for the CRISPR/Cas9 System for Model Organism Genomes

2013 ◽  
Vol 2013 ◽  
pp. 1-4 ◽  
Author(s):  
Ming Ma ◽  
Adam Y. Ye ◽  
Weiguo Zheng ◽  
Lei Kong
Keyword(s):  
Rna Secondary Structure ◽  
Gene Disruption ◽  
Model Organism ◽  
Eukaryotic Cells ◽  
Model Organisms ◽  
Rna Sequences ◽  
Sequence Design ◽  
Rna Sequence ◽  
Guide Rna ◽  
Targeted Gene Disruption

Cas9/CRISPR has been reported to efficiently induce targeted gene disruption and homologous recombination in both prokaryotic and eukaryotic cells. Thus, we developed a Guide RNA Sequence Design Platform for the Cas9/CRISPR silencing system for model organisms. The platform is easy to use for gRNA design with input query sequences. It finds potential targets by PAM and ranks them according to factors including uniqueness, SNP, RNA secondary structure, and AT content. The platform allows users to upload and share their experimental results. In addition, most guide RNA sequences from published papers have been put into our database.

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 dbGuide: a database of functionally validated guide RNAs for genome editing in human and mouse cells

2020 ◽  
Vol 49 (D1) ◽  
pp. D871-D876
Author(s):  
Alexander A Gooden ◽  
Christine N Evans ◽  
Timothy P Sheets ◽  
Michelle E Clapp ◽  
Raj Chari
Keyword(s):  
Epigenetic Modification ◽  
Gene Activation ◽  
Amplicon Sequencing ◽  
Ease Of Use ◽  
Rna Sequences ◽  
Additional Species ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Human And Mouse

Abstract With the technology's accessibility and ease of use, CRISPR has been employed widely in many different organisms and experimental settings. As a result, thousands of publications have used CRISPR to make specific genetic perturbations, establishing in itself a resource of validated guide RNA sequences. While numerous computational tools to assist in the design and identification of candidate guide RNAs exist, these are still just at best predictions and generally, researchers inevitably will test multiple sequences for functional activity. Here, we present dbGuide (https://sgrnascorer.cancer.gov/dbguide), a database of functionally validated guide RNA sequences for CRISPR/Cas9-based knockout in human and mouse. Our database not only contains computationally determined candidate guide RNA sequences, but of even greater value, over 4000 sequences which have been functionally validated either through direct amplicon sequencing or manual curation of literature from over 1000 publications. Finally, our established framework will allow for continual addition of newly published and experimentally validated guide RNA sequences for CRISPR/Cas9-based knockout as well as incorporation of sequences from different gene editing systems, additional species and other types of site-specific functionalities such as base editing, gene activation, repression and epigenetic modification.

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