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.

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 Moreover, online tools are limited to a restricted list of reference genome and lack the flexibility to incorporate unpublished genomes or contemplate genomes of populations with allelic variants.ResultsTo address these issues, I have developed the ARES-GT, a local command line tool in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 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 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 Structural basis for Cas9 off-target activity

2021 ◽  
Author(s):  
Martin Pacesa ◽  
Chun-Han Lin ◽  
Antoine Clery ◽  
Katja Bargsten ◽  
Matthew J. Irby ◽  
...  
Keyword(s):  
Rational Design ◽  
Target Prediction ◽  
Structural Basis ◽  
Guide Rna ◽  
Guide Rnas ◽  
Target Dna ◽  
Bona Fide ◽  
Dna Specificity ◽  
Target Binding ◽  
Target Activity

The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of non- canonical base pairing interactions and preservation of base stacking within the guide-off-target heteroduplex. Off-target sites containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping rather than RNA bulge formation. Additionally, PAM-distal mismatches result in duplex unpairing and induce a conformational change of the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.

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 Nucleolar Factors Direct the 2′-O-Ribose Methylation and Pseudouridylation of U6 Spliceosomal RNA

1999 ◽  
Vol 19 (10) ◽  
pp. 6906-6917 ◽  
Author(s):  
Philippe Ganot ◽  
Beáta E. Jády ◽  
Marie-Line Bortolin ◽  
Xavier Darzacq ◽  
Tamás Kiss
Keyword(s):  
Small Nuclear Rna ◽  
Guide Rna ◽  
Guide Rnas ◽  
Trafficking Pathway ◽  
5.8S Rrna ◽  
Nuclear Rna ◽  
Nucleolar Rna ◽  
Correct Target ◽  
Selection Of ◽  
U6 Rna

ABSTRACT The nucleolus has long been known as a functionally highly specialized subnuclear compartment where synthesis, posttranscriptional modification, and processing of cytoplasmic rRNAs take place. In this study, we demonstrate that the nucleolus contains all thetrans-acting factors that are responsible for the accurate and efficient synthesis of the eight 2′-O-methylated nucleotides and three pseudouridine residues carried by the mammalian U6 spliceosomal small nuclear RNA. Factors mediating the formation of pseudouridine residues in the U3 small nucleolar RNA are also present and functionally active in the nucleolus. For selection of the correct target nucleotides in the U6 and U3 RNAs, the nucleolar 2′-O-methylation and pseudouridylation factors rely on short sequences located around the target nucleotide to be modified. This observation further underscores a recently proposed role for small nucleolar guide RNAs in the 2′-O-methylation of the U6 spliceosomal RNA (K. T. Tycowski, Z.-H. You, P. J. Graham, and J. A. Steitz, Mol. Cell 2:629–638, 1998). We demonstrate that a novel 2′-O-methylated nucleotide can be generated in the yeast U6 RNA by use of an artificial 2′-O-methylation small nucleolar guide RNA. We also show that a short fragment of the 5.8S rRNA, when expressed as part of the human U6 RNA, is faithfully 2′-O-methylated and pseudouridylated. These results are most consistent with a trafficking pathway in which the U6 spliceosomal RNA cycles through the nucleolus to undergo nucleolar RNA-directed modifications.

scholarly journals Evaluation and rational design of guide RNAs for efficient CRISPR/Cas9-mediated mutagenesis in Ciona

2016 ◽  
Author(s):  
Shashank Gandhi ◽  
Maximilian Haeussler ◽  
Florian Razy-Krajka ◽  
Lionel Christiaen ◽  
Alberto Stolfi
Keyword(s):  
High Throughput ◽  
Rational Design ◽  
Genome Engineering ◽  
High Throughput Sequencing ◽  
Loss Of Function ◽  
Tissue Specific ◽  
Guide Rna ◽  
Guide Rnas ◽  
Highly Active

AbstractThe CRISPR/Cas9 system has emerged as an important tool for various genome engineering applications. A current obstacle to high throughput applications of CRISPR/Cas9 is the imprecise prediction of highly active single guide. RNAs (sgRNAs). We previously implemented the CRISPR/Cas9 system to induce tissue-specific mutations in the tunicate Ciona. In the present study, we designed and tested 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues of Ciona embryo. Using high-throughput sequencing of mutagenized alleles, we identified guide sequences that correlate with sgRNA mutagenesis activity and used this information for the rational design of all possible sgRNAs targeting the Ciona transcriptome. We also describe a one-step cloning-free protocol for the assembly of sgRNA expression cassettes. These cassettes can be directly electroporated as unpurified PCR products into Ciona embryos for sgRNA expression in vivo, resulting in high frequency of CRISPR/Cas9-mediated mutagenesis in somatic cells of electroporated embryos.We found a strong correlation between the frequency of an Ebf loss-of-function phenotype and the mutagenesis efficacies of individual Ebf-targeting sgRNAs tested using this method. We anticipate that our approach can be scaled up to systematically design and deliver highly efficient sgRNAs for the tissue-specific investigation of gene functions in Ciona.

Apoptosis and development

2003 ◽  
Vol 39 ◽  
pp. 11-24 ◽  
Author(s):  
Justin V McCarthy
Keyword(s):  
Drosophila Melanogaster ◽  
Mammalian Cells ◽  
Cell Number ◽  
Model Organisms ◽  
Biological Processes ◽  
Nematode Caenorhabditis Elegans ◽  
Apoptotic Pathway ◽  
Genetic Pathways ◽  
Evolutionarily Conserved ◽  
Multicellular Organisms

Apoptosis is an evolutionarily conserved process used by multicellular organisms to developmentally regulate cell number or to eliminate cells that are potentially detrimental to the organism. The large diversity of regulators of apoptosis in mammalian cells and their numerous interactions complicate the analysis of their individual functions, particularly in development. The remarkable conservation of apoptotic mechanisms across species has allowed the genetic pathways of apoptosis determined in lower species, such as the nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster, to act as models for understanding the biology of apoptosis in mammalian cells. Though many components of the apoptotic pathway are conserved between species, the use of additional model organisms has revealed several important differences and supports the use of model organisms in deciphering complex biological processes such as apoptosis.

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

2020 ◽  
Author(s):  
Y.V. Mikhaylova ◽  
◽  
M.A. Tyumentseva ◽  
A.A. Shelenkov ◽  
Y.G. Yanushevich ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Correct Choice ◽  
Target Sequence ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Chemokine Receptor Ccr5 ◽  
Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

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