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 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 ATTfold: RNA Secondary Structure Prediction With Pseudoknots Based on Attention Mechanism

2020 ◽  
Vol 11 ◽  
Author(s):  
Yili Wang ◽  
Yuanning Liu ◽  
Shuo Wang ◽  
Zhen Liu ◽  
Yubing Gao ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Structure Prediction ◽  
Rna Secondary Structure ◽  
Secondary Structure Prediction ◽  
Attention Mechanism ◽  
Soft Constraints ◽  
Rna Sequences ◽  
Rna Secondary Structure Prediction ◽  
Rna Sequence ◽  
Structure Information

Accurate RNA secondary structure information is the cornerstone of gene function research and RNA tertiary structure prediction. However, most traditional RNA secondary structure prediction algorithms are based on the dynamic programming (DP) algorithm, according to the minimum free energy theory, with both hard and soft constraints. The accuracy is particularly dependent on the accuracy of soft constraints (from experimental data like chemical and enzyme detection). With the elongation of the RNA sequence, the time complexity of DP-based algorithms will increase geometrically, as a result, they are not good at coping with relatively long sequences. Furthermore, due to the complexity of the pseudoknots structure, the secondary structure prediction method, based on traditional algorithms, has great defects which cannot predict the secondary structure with pseudoknots well. Therefore, few algorithms have been available for pseudoknots prediction in the past. The ATTfold algorithm proposed in this article is a deep learning algorithm based on an attention mechanism. It analyzes the global information of the RNA sequence via the characteristics of the attention mechanism, focuses on the correlation between paired bases, and solves the problem of long sequence prediction. Moreover, this algorithm also extracts the effective multi-dimensional features from a great number of RNA sequences and structure information, by combining the exclusive hard constraints of RNA secondary structure. Hence, it accurately determines the pairing position of each base, and obtains the real and effective RNA secondary structure, including pseudoknots. Finally, after training the ATTfold algorithm model through tens of thousands of RNA sequences and their real secondary structures, this algorithm was compared with four classic RNA secondary structure prediction algorithms. The results show that our algorithm significantly outperforms others and more accurately showed the secondary structure of RNA. As the data in RNA sequence databases increase, our deep learning-based algorithm will have superior performance. In the future, this kind of algorithm will be more indispensable.

scholarly journals In Search of Species-Specific SNPs in a Non-Model Animal (European Bison (Bison bonasus))—Comparison of De Novo and Reference-Based Integrated Pipeline of STACKS Using Genotyping-by-Sequencing (GBS) Data

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2226
Author(s):  
Sazia Kunvar ◽  
Sylwia Czarnomska ◽  
Cino Pertoldi ◽  
Małgorzata Tokarska
Keyword(s):  
Reference Genome ◽  
De Novo ◽  
Bos Taurus ◽  
Model Organism ◽  
Genotyping By Sequencing ◽  
Model Organisms ◽  
European Bison ◽  
Model Animal ◽  
Pcr Duplicates ◽  
Species Specific

The European bison is a non-model organism; thus, most of its genetic and genomic analyses have been performed using cattle-specific resources, such as BovineSNP50 BeadChip or Illumina Bovine 800 K HD Bead Chip. The problem with non-specific tools is the potential loss of evolutionary diversified information (ascertainment bias) and species-specific markers. Here, we have used a genotyping-by-sequencing (GBS) approach for genotyping 256 samples from the European bison population in Bialowieza Forest (Poland) and performed an analysis using two integrated pipelines of the STACKS software: one is de novo (without reference genome) and the other is a reference pipeline (with reference genome). Moreover, we used a reference pipeline with two different genomes, i.e., Bos taurus and European bison. Genotyping by sequencing (GBS) is a useful tool for SNP genotyping in non-model organisms due to its cost effectiveness. Our results support GBS with a reference pipeline without PCR duplicates as a powerful approach for studying the population structure and genotyping data of non-model organisms. We found more polymorphic markers in the reference pipeline in comparison to the de novo pipeline. The decreased number of SNPs from the de novo pipeline could be due to the extremely low level of heterozygosity in European bison. It has been confirmed that all the de novo/Bos taurus and Bos taurus reference pipeline obtained SNPs were unique and not included in 800 K BovineHD BeadChip.

scholarly journals Alliance of Genome Resources Portal: unified model organism research platform

2019 ◽  
Vol 48 (D1) ◽  
pp. D650-D658 ◽  
Author(s):  
◽  
Julie Agapite ◽  
Laurent-Philippe Albou ◽  
Suzi Aleksander ◽  
Joanna Argasinska ◽  
...  
Keyword(s):  
Gene Ontology ◽  
Model Organism ◽  
Model Organisms ◽  
Data Types ◽  
Primary Model ◽  
Genomic Studies ◽  
Health And Disease ◽  
Extensive Body ◽  
Access To Data ◽  
Model Organism Databases

Abstract The Alliance of Genome Resources (Alliance) is a consortium of the major model organism databases and the Gene Ontology that is guided by the vision of facilitating exploration of related genes in human and well-studied model organisms by providing a highly integrated and comprehensive platform that enables researchers to leverage the extensive body of genetic and genomic studies in these organisms. Initiated in 2016, the Alliance is building a central portal (www.alliancegenome.org) for access to data for the primary model organisms along with gene ontology data and human data. All data types represented in the Alliance portal (e.g. genomic data and phenotype descriptions) have common data models and workflows for curation. All data are open and freely available via a variety of mechanisms. Long-term plans for the Alliance project include a focus on coverage of additional model organisms including those without dedicated curation communities, and the inclusion of new data types with a particular focus on providing data and tools for the non-model-organism researcher that support enhanced discovery about human health and disease. Here we review current progress and present immediate plans for this new bioinformatics resource.

scholarly journals Transgenic Epigenetics: Using Transgenic Organisms to Examine Epigenetic Phenomena

2012 ◽  
Vol 2012 ◽  
pp. 1-14 ◽  
Author(s):  
Lori A. McEachern
Keyword(s):  
Molecular Mechanisms ◽  
Model Organism ◽  
Evolutionary Conservation ◽  
Model Organisms ◽  
Valuable Insight ◽  
Epigenetic Control ◽  
Transgenic Organisms ◽  
Epigenetic Analysis ◽  
Insight Into ◽  
Epigenetic Processes

Non-model organisms are generally more difficult and/or time consuming to work with than model organisms. In addition, epigenetic analysis of model organisms is facilitated by well-established protocols, and commercially-available reagents and kits that may not be available for, or previously tested on, non-model organisms. Given the evolutionary conservation and widespread nature of many epigenetic mechanisms, a powerful method to analyze epigenetic phenomena from non-model organisms would be to use transgenic model organisms containing an epigenetic region of interest from the non-model. Interestingly, while transgenic Drosophila and mice have provided significant insight into the molecular mechanisms and evolutionary conservation of the epigenetic processes that target epigenetic control regions in other model organisms, this method has so far been under-exploited for non-model organism epigenetic analysis. This paper details several experiments that have examined the epigenetic processes of genomic imprinting and paramutation, by transferring an epigenetic control region from one model organism to another. These cross-species experiments demonstrate that valuable insight into both the molecular mechanisms and evolutionary conservation of epigenetic processes may be obtained via transgenic experiments, which can then be used to guide further investigations and experiments in the species of interest.

scholarly journals Nonhuman primates’ tissue banks: resources for all model organism research

2021 ◽  
Author(s):  
Claire Witham ◽  
Sara Wells
Keyword(s):  
Nonhuman Primates ◽  
Ex Vivo ◽  
Model Organism ◽  
Translation Studies ◽  
Model Organisms ◽  
Laboratory Animals ◽  
Tissue Banks ◽  
Post Mortem ◽  
Wide Range ◽  
Research Programmes

AbstractBiobanks containing tissue and other biological samples from many model organisms provide easy and faster access to ex vivo resources for a wide-range of research programmes. For all laboratory animals, collecting and preserving tissue at post-mortem is an effective way of maximising the benefits of individual animals and potentially reducing the numbers required for experimentation in the future. For primate tissues, biobanks represent the scarcest of these resources but quite possibly those most valuable for preclinical and translation studies.

scholarly journals Development of a targeted gene disruption system in the PET-degrading bacterium Ideonella sakaiensis and its applications to PETase and MHETase genes

2021 ◽  
Author(s):  
Shin-ichi Hachisuka ◽  
Tarou Nishii ◽  
Shosuke Yoshida
Keyword(s):  
Terephthalic Acid ◽  
Parent Strain ◽  
Gene Disruption ◽  
Ethylene Terephthalate ◽  
Targeted Gene Disruption ◽  
Degrading Bacterium ◽  
Poly Ethylene Terephthalate ◽  
Poly Ethylene ◽  
Pet Hydrolase

Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however its non-biodegradability results in a large amount of waste accumulation that has a negative impact on the environment. Recently, a PET-degrading bacterium Ideonella sakaiensis 201-F6 strain was isolated and the enzymes involved in PET-digestion, PET hydrolase (PETase) and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase (MHETase), were identified. Despite the great potentials of I. sakaiensis in bioremediation and biorecycling, approaches to studying this bacterium remain limited. In this study, to enable the functional analysis of PETase and MHETase genes in vivo , we have developed a gene disruption system in I. sakaiensis . The pT18 mobsacB -based disruption vector harboring directly connected 5'- and 3'-flanking regions of the target gene for homologous recombination was introduced into I. sakaiensis cells via conjugation. First, we deleted the orotidine 5'-phosphate decarboxylase gene ( pyrF ) from the genome of the wild-type strain, producing the Δ pyrF strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the Δ pyrF strain as a parent strain, and pyrF as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both Δ petase and Δ mhetase strains on terephthalic acid (TPA, one of the PET hydrolytic products) was comparable to that of the parent strain. However, these mutant strains dramatically decreased the growth level on PET to that on no carbon source. Moreover, the Δ petase strain completely abolished PET degradation capacity. These results demonstrate that PETase and MHETase are essential for I. sakaiensis metabolism of PET. IMPORTANCE The poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis possesses two unique enzymes able to serve in PET hydrolysis. PET hydrolase (PETase) hydrolyzes PET into mono(2-hydroxyethyl) terephthalic acid (MHET) and MHET hydrolase (MHETase) hydrolyzes MHET into terephthalic acid (TPA) and ethylene glycol (EG). These enzymes have attracted global attention as they have potential to be used for bioconversion of PET. Compared to many in vitro studies including the biochemical and crystal structure analyses, few in vivo studies have been reported. Here, we developed a targeted gene disruption system in I. sakaiensis , which was then applied for constructing Δ petase and Δ mhetase strains. Growth of these disruptants revealed that PETase is a sole enzyme responsible for PET degradation in I. sakaiensis , while PETase and MHETase play essential roles in its PET assimilation.

scholarly journals Accurate allele frequencies from ultra-low coverage pool-seq samples in evolve-and-resequence experiments

2018 ◽  
Author(s):  
Susanne Tilk ◽  
Alan Bergland ◽  
Aaron Goodman ◽  
Paul Schmidt ◽  
Dmitri Petrov ◽  
...  
Keyword(s):  
Allele Frequency ◽  
Model Organism ◽  
Software Tool ◽  
Allele Frequencies ◽  
Model Organisms ◽  
Sequencing Data ◽  
High Coverage ◽  
Next Generation Sequencing Technology ◽  
Low Coverage ◽  
Pooled Samples

AbstractEvolve-and-resequence (E+R) experiments leverage next-generation sequencing technology to track the allele frequency dynamics of populations as they evolve. While previous work has shown that adaptive alleles can be detected by comparing frequency trajectories from many replicate populations, this power comes at the expense of high-coverage (>100x) sequencing of many pooled samples, which can be cost-prohibitive. Here, we show that accurate estimates of allele frequencies can be achieved with very shallow sequencing depths (<5x) via inference of known founder haplotypes in small genomic windows. This technique can be used to efficiently estimate frequencies for any number of bi-allelic SNPs in populations of any model organism founded with sequenced homozygous strains. Using both experimentally-pooled and simulated samples of Drosophila melanogaster, we show that haplotype inference can improve allele frequency accuracy by orders of magnitude for up to 50 generations of recombination, and is robust to moderate levels of missing data, as well as different selection regimes. Finally, we show that a simple linear model generated from these simulations can predict the accuracy of haplotype-derived allele frequencies in other model organisms and experimental designs. To make these results broadly accessible for use in E+R experiments, we introduce HAF-pipe, an open-source software tool for calculating haplotype-derived allele frequencies from raw sequencing data. Ultimately, by reducing sequencing costs without sacrificing accuracy, our method facilitates E+R designs with higher replication and resolution, and thereby, increased power to detect adaptive alleles.

scholarly journals Targeted Gene Disruption in Zebrafish Reveals Noncanonical Functions of LH Signaling in Reproduction

2014 ◽  
Vol 28 (11) ◽  
pp. 1785-1795 ◽  
Author(s):  
Lianhe Chu ◽  
Jianzhen Li ◽  
Yun Liu ◽  
Wei Hu ◽  
Christopher H. K. Cheng
Keyword(s):  
Gene Disruption ◽  
Targeted Gene Disruption
Sign in / Sign up

Export Citation Format

Share Document