scholarly journals Scalable Design of paired CRISPR Guide RNAs for Genomic Deletion

2016 ◽  
Author(s):  
Carlos Pulido-Quetglas ◽  
Estel Aparicio-Prat ◽  
Carme Arnan ◽  
Taisia Polidori ◽  
Toni Hermoso ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Model Organisms ◽  
High Coverage ◽  
Bioinformatic Pipeline ◽  
Genomic Deletion ◽  
C Elegans ◽  
Guide Rnas ◽  
Single Target ◽  
Sgrna Design ◽  
Scalable Design

AbstractUsing CRISPR/Cas9, diverse genomic elements may be studied in their endogenous context. Pairs of single guide RNAs (sgRNAs) are used to delete regulatory elements and small RNA genes, while longer RNAs can be silenced through promoter deletion. We here present CRISPETa, a bioinformatic pipeline for flexible and scalable paired sgRNA design based on an empirical scoring model. Multiple sgRNA pairs are returned for each target. Any number of targets can be analyzed in parallel, making CRISPETa equally appropriate for studies of individual elements, or complex library screens. Fast run-times are achieved using a precomputed off-target database. sgRNA pair designs are output in a convenient format for visualisation and oligonucleotide ordering. We present a series of pre-designed, high-coverage library designs for entire classes of non-coding elements in human, mouse, zebrafish, Drosophila and C. elegans. Using an improved version of the DECKO deletion vector, together with a quantitative deletion assay, we test CRISPETa designs by deleting an enhancer and exonic fragment of the MALAT1 oncogene. These achieve efficiencies of ≥50%, resulting in production of mutant RNA. CRISPETa will be useful for researchers seeking to harness CRISPR for targeted genomic deletion, in a variety of model organisms, from single-target to high-throughput scales.

scholarly journals Scalable Design of Paired CRISPR Guide RNAs for Genomic Deletion

2017 ◽  
Vol 13 (3) ◽  
pp. e1005341 ◽  
Author(s):  
Carlos Pulido-Quetglas ◽  
Estel Aparicio-Prat ◽  
Carme Arnan ◽  
Taisia Polidori ◽  
Toni Hermoso ◽  
...  
Keyword(s):  
Genomic Deletion ◽  
Guide Rnas ◽  
Scalable Design

scholarly journals Parallel genetics of regulatory sequences in vivo

2020 ◽  
Author(s):  
Jonathan Froehlich ◽  
Bora Uyar ◽  
Margareta Herzog ◽  
Kathrin Theil ◽  
Petar Glažar ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Phenotypic Traits ◽  
Regulatory Sequences ◽  
C Elegans ◽  
Guide Rnas ◽  
Sequence Elements ◽  
Sequencing Strategy ◽  
Genomic Environment ◽  
Reporter Assays

AbstractUnderstanding how regulatory sequences control gene expression is fundamental to explain how phenotypes arise in health and disease. Traditional reporter assays inform about function of individual regulatory elements, typically in isolation. However, regulatory elements must ultimately be understood by perturbing them within their genomic environment and developmental- or tissue-specific contexts. This is technically challenging; therefore, few regulatory elements have been characterized in vivo. Here, we used inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3′ UTRs of 16 genes in C. elegans. To quantify the consequences of mutations on expression, we developed a targeted RNA sequencing strategy across hundreds of mutant animals. We were also able to systematically and quantitatively assign fitness cost to mutations. Finally, we identified and characterized sequence elements that strongly regulate phenotypic traits. Our approach enables highly parallelized, functional analysis of regulatory sequences in vivo.

The glycomes of Caenorhabditis elegans and other model organisms

2002 ◽  
Vol 69 ◽  
pp. 117-134 ◽  
Author(s):  
Stuart M. Haslam ◽  
David Gems ◽  
Howard R. Morris ◽  
Anne Dell
Keyword(s):  
Caenorhabditis Elegans ◽  
Current Knowledge ◽  
Structural Data ◽  
Model Organisms ◽  
Entire Genome ◽  
C Elegans ◽  
The Face ◽  
Area Of Concern ◽  
Nematode Worm

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fucα1–2Gal1–2Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.

scholarly journals Myosinome: A Database of Myosins from Select Eukaryotic Genomes to Facilitate Analysis of Sequence-Structure-Function Relationships

2012 ◽  
Vol 6 ◽  
pp. BBI.S9902 ◽  
Author(s):  
Divya P. Syamaladevi ◽  
Margaret S Sunitha ◽  
S. Kalaimathy ◽  
Chandrashekar C. Reddy ◽  
Mohammed Iftekhar ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Homo Sapiens ◽  
Relevant Literature ◽  
Myosin Ii ◽  
Coiled Coil ◽  
Structural Features ◽  
Model Organisms ◽  
Congenital Diseases ◽  
C Elegans

Myosins are one of the largest protein superfamilies with 24 classes. They have conserved structural features and catalytic domains yet show huge variation at different domains resulting in a variety of functions. Myosins are molecules driving various kinds of cellular processes and motility until the level of organisms. These are ATPases that utilize the chemical energy released by ATP hydrolysis to bring about conformational changes leading to a motor function. Myosins are important as they are involved in almost all cellular activities ranging from cell division to transcriptional regulation. They are crucial due to their involvement in many congenital diseases symptomatized by muscular malfunctions, cardiac diseases, deafness, neural and immunological dysfunction, and so on, many of which lead to death at an early age. We present Myosinome, a database of selected myosin classes (myosin II, V, and VI) from five model organisms. This knowledge base provides the sequences, phylogenetic clustering, domain architectures of myosins and molecular models, structural analyses, and relevant literature of their coiled-coil domains. In the current version of Myosinome, information about 71 myosin sequences belonging to three myosin classes (myosin II, V, and VI) in five model organisms ( Homo Sapiens, Mus musculus, D. melanogaster, C. elegans and S. cereviseae) identified using bioinformatics surveys are presented, and several of them are yet to be functionally characterized. As these proteins are involved in congenital diseases, such a database would be useful in short-listing candidates for gene therapy and drug development. The database can be accessed from http://caps.ncbs.res.in/myosinome .

scholarly journals Multi-environment phenotyping of C. elegans for robust evaluation of physical performance

2020 ◽  
Author(s):  
Jennifer E. Hewitt ◽  
Ricardo Laranjeiro ◽  
Masoud Norouzi ◽  
Rebecca Ellwood ◽  
Adam Antebi ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Physical Performance ◽  
Current Treatment ◽  
Therapeutic Interventions ◽  
Model Organisms ◽  
C Elegans ◽  
Drug Candidates ◽  
Treatment Standard ◽  
Drug Interventions

ABSTRACTDetermining the physical performance of humans using several measures is essential to evaluating the severity of diseases, understanding the role of environmental factors, and developing therapeutic interventions. Development of analogous measures of physical performance in model organisms can help in identifying conserved signaling pathways and prioritizing drug candidates. In this study, we propose a multi-environment phenotyping (MEP) approach that generates a comprehensive set of measures indicative of physical performance in C. elegans. We challenge C. elegans in different mechanical environments of burrowing, swimming, and crawling, each of which places different physiological demands on the animals to generate locomotory forces. Implementation of the MEP approach is done using three established assays corresponding to each environment–a hydrogel-based burrowing assay, the CeleST swim assay, and the NemaFlex crawling strength assay. Using this approach, we study individuals and show that these three assays report on unique aspects of nematode physiology, as phenotypic measures obtained from different environments do not correlate with one another. Analysis of a subset of genes representative of oxidative stress, glucose metabolism, and fat metabolism show differential expression depending on the animal’s environment, suggesting that each environment evokes a response with distinct genetic requirements. To demonstrate the utility of the MEP platform, we evaluate the response of a muscular dystrophy model of C. elegans dys-1 to drug interventions of prednisone, melatonin and serotonin. We find that prednisone, which is the current treatment standard for human Duchenne muscular dystrophy, confers benefits in all three assays. Furthermore, while the tested compounds improve the physical performance of dys-1, these compounds are not able to fully restore the measures to wild-type levels, suggesting the need for discovery efforts to identify more efficacious compounds that could be aided using the MEP platform. In summary, the MEP platform’s ability to robustly define C. elegans locomotory phenotypes demonstrates the utility of the MEP approach toward identification of candidates for therapeutic intervention, especially in disease models in which the neuromuscular performance is impaired.

scholarly journals Identification of high-efficiency 3′GG gRNA motifs in indexed FASTA files with ngg2

2015 ◽  
Vol 1 ◽  
pp. e33 ◽  
Author(s):  
Elisha D. Roberson
Keyword(s):  
High Efficiency ◽  
Homo Sapiens ◽  
Model Organisms ◽  
Proof Of Concept ◽  
Protein Coding ◽  
C Elegans ◽  
Protein Coding Genes ◽  
Starting Point ◽  
Command Line Tool ◽  
Reference Genomes

CRISPR/Cas9 is emerging as one of the most-used methods of genome modification in organisms ranging from bacteria to human cells. However, the efficiency of editing varies tremendously site-to-site. A recent report identified a novel motif, called the 3′GG motif, which substantially increases the efficiency of editing at all sites tested inC. elegans. Furthermore, they highlighted that previously published gRNAs with high editing efficiency also had this motif. I designed a Python command-line tool, ngg2, to identify 3′GG gRNA sites from indexed FASTA files. As a proof-of-concept, I screened for these motifs in six model genomes:Saccharomyces cerevisiae,Caenorhabditis elegans,Drosophila melanogaster,Danio rerio,Mus musculus, andHomo sapiens. I also scanned the genomes of pig (Sus scrofa) and African elephant (Loxodonta africana) to demonstrate the utility in non-model organisms. I identified more than 60 million single match 3′GG motifs in these genomes. Greater than 61% of all protein coding genes in the reference genomes had at least one unique 3′GG gRNA site overlapping an exon. In particular, more than 96% of mouse and 93% of human protein coding genes have at least one unique, overlapping 3′GG gRNA. These identified sites can be used as a starting point in gRNA selection, and the ngg2 tool provides an important ability to identify 3′GG editing sites in any species with an available genome sequence.

scholarly journals Notch-dependent DNA cis-regulatory elements and their dose-dependent control of C. elegans stem cell self-renewal

2021 ◽  
Author(s):  
Tina R. Lynch ◽  
Mingyu Xue ◽  
Cazza W. Czerniak ◽  
ChangHwan Lee ◽  
Judith Kimble
Keyword(s):  
Stem Cell ◽  
Regulatory Elements ◽  
Single Element ◽  
Developmental Context ◽  
C Elegans ◽  
Stem Cell Maintenance ◽  
Nascent Transcripts ◽  
Functional Analyses ◽  
Dose Dependent

A long-standing biological question is how DNA cis-regulatory elements shape transcriptional patterns during metazoan development. The use of reporter constructs, cell culture and computational modeling has made enormous contributions to understanding this fundamental question, but analysis of regulatory elements in their natural developmental context is an essential but rarely used complement. Here, we edited Notch-dependent cis-regulatory elements in the endogenous C. elegans sygl-1 gene, which encodes a key stem cell regulator. We then analyzed the in vivo consequences of those mutations – on both gene expression (nascent transcripts, mRNA, protein) and stem cell maintenance. Mutation of a single element in a three-element homotypic cluster reduced expression as well as stem cell pool size by about half, while mutation of two elements essentially abolished them. We find that LBS number and LBS neighborhood are both important to activity: elements on separate chromosomes function additively, while elements in the same cluster act synergistically. Our approach of precise CRISPR/Cas9 gene editing coupled with quantitation of both molecular and biological readouts establishes a powerful model for in vivo functional analyses of DNA cis-regulatory elements.

scholarly journals Chromatin accessibility is dynamically regulated across C. elegans development and ageing

2018 ◽  
Author(s):  
Jürgen Jänes ◽  
Yan Dong ◽  
Michael Schoof ◽  
Jacques Serizay ◽  
Alex Appert ◽  
...  
Keyword(s):  
Regulatory Mechanism ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Protein Coding ◽  
C Elegans ◽  
Transcription Profiles ◽  
Physiological Processes ◽  
Global Identification ◽  
Identification And Characterization

AbstractAn essential step for understanding the transcriptional circuits that control development and physiology is the global identification and characterization of regulatory elements. Here we present the first map of regulatory elements across the development and ageing of an animal, identifying 42,245 elements accessible in at least one C. elegans stage. Based on nuclear transcription profiles, we define 15,714 protein-coding promoters and 19,231 putative enhancers, and find that both types of element can drive orientation-independent transcription. Additionally, hundreds of promoters produce transcripts antisense to protein coding genes, suggesting involvement in a widespread regulatory mechanism. We find that the accessibility of most elements is regulated during development and/or ageing and that patterns of accessibility change are linked to specific developmental or physiological processes. The map and characterization of regulatory elements across C. elegans life provides a platform for understanding how transcription controls development and ageing.

scholarly journals Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhiwei Hu ◽  
Yannan Wang ◽  
Qian Liu ◽  
Yan Qiu ◽  
Zhiyu Zhong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Target Sites ◽  
Guide Sequence ◽  
Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

scholarly journals C. elegans neurons have functional dendritic spines

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Andrea Cuentas-Condori ◽  
Ben Mulcahy ◽  
Siwei He ◽  
Sierra Palumbos ◽  
Mei Zhen ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Dendritic Spines ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Live Cell ◽  
Model Organisms ◽  
Spine Density ◽  
C Elegans ◽  
Spine Function

Dendritic spines are specialized postsynaptic structures that transduce presynaptic signals, are regulated by neural activity and correlated with learning and memory. Most studies of spine function have focused on the mammalian nervous system. However, spine-like protrusions have been reported in C. elegans (Philbrook et al., 2018), suggesting that the experimental advantages of smaller model organisms could be exploited to study the biology of dendritic spines. Here, we used super-resolution microscopy, electron microscopy, live-cell imaging and genetics to show that C. elegans motor neurons have functional dendritic spines that: (1) are structurally defined by a dynamic actin cytoskeleton; (2) appose presynaptic dense projections; (3) localize ER and ribosomes; (4) display calcium transients triggered by presynaptic activity and propagated by internal Ca++ stores; (5) respond to activity-dependent signals that regulate spine density. These studies provide a solid foundation for a new experimental paradigm that exploits the power of C. elegans genetics and live-cell imaging for fundamental studies of dendritic spine morphogenesis and function.

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