scholarly journals Pipeline for generating stable large genomic deletions in zebrafish, from small domains to whole gene excisions

2021 ◽  
Author(s):  
Alisha Tromp ◽  
Kate Robinson ◽  
Thomas E. Hall ◽  
Bryan Mowry ◽  
Jean Giacomotto
Keyword(s):  
Large Deletion ◽  
Genomic Region ◽  
Base Pairs ◽  
Genomic Locus ◽  
Transmission Frequency ◽  
Genomic Deletions ◽  
Large Deletions ◽  
Multiple Promoters ◽  
Target Sites ◽  
Genomic Regions

ABSTRACTHere we describe a short feasibility study and methodological framework for the production of stable, CRISPR/Cas9-based, large genomic deletions in zebrafish, ranging from several base pairs (bp) to hundreds of kilobases (kb). Using a cocktail of four sgRNAs targeting a single genomic region mixed with a marker-sgRNA against the pigmentation gene tyrosinase (tyr), we demonstrate that one can easily and accurately excise genomic regions such as promoters, protein domains, specific exons or whole genes. We exemplify this technique with a complex gene family, neurexins, composed of three duplicated genes with multiple promoters and intricate splicing processes leading to thousands of isoforms. We precisely deleted small regions such as their transmembrane domains (150bp deletion in average) to their entire genomic locus (300kb deletion for nrxn1A for instance). We find that both the concentration and ratio of Cas9/sgRNAs are critical for the successful generation of these large deletions and, interestingly, that their transmission frequency does not decrease with increasing distance between sgRNA target sites. Considering the growing reports and debate about genetically compensated small indel mutants, the use of large-deletion approaches is likely to be widely adopted in studies of gene function. This strategy will also be key to the study of non-coding genomic regions.

scholarly journals Pipeline for generating stable large genomic deletions in zebrafish, from small domains to whole gene excisions

2021 ◽  
Author(s):  
Alisha Tromp ◽  
Kate Robinson ◽  
Thomas E Hall ◽  
Bryan Mowry ◽  
Jean Giacomotto
Keyword(s):  
Large Deletion ◽  
Genomic Region ◽  
Base Pairs ◽  
Genomic Locus ◽  
Transmission Frequency ◽  
Genomic Deletions ◽  
Large Deletions ◽  
Multiple Promoters ◽  
Target Sites ◽  
Genomic Regions

Abstract Here we describe a short feasibility study and methodological framework for the production of stable, CRISPR/Cas9-based, large genomic deletions in zebrafish, ranging from several base pairs (bp) to hundreds of kilobases (kb). Using a cocktail of four sgRNAs targeting a single genomic region mixed with a marker-sgRNA against the pigmentation gene tyrosinase (tyr), we demonstrate that one can easily and accurately excise genomic regions such as promoters, protein domains, specific exons or whole genes. We exemplify this technique with a complex gene family, neurexins, composed of three duplicated genes with multiple promoters and intricate splicing processes leading to thousands of isoforms. We precisely deleted small regions such as their transmembrane domains (150 bp deletion in average) to their entire genomic locus (300 kb deletion for nrxn1a for instance). We find that both the concentration and ratio of Cas9/sgRNAs are critical for the successful generation of these large deletions and, interestingly, that in our study their transmission frequency does not seem to decrease with increasing distance between sgRNA target sites. Considering the growing reports and debate about genetically compensated small indel mutants, the use of large-deletion approaches is likely to be widely adopted in studies of gene function. This strategy will also be key to the study of non-coding genomic regions. Note that we are also describing here a custom method to produce the sgRNAs, which proved to be faster and more robust than the ones traditionally used in the community to date.

scholarly journals Molecular and Functional Mapping of the Piebald Deletion Complex on Mouse Chromosome 14

Genetics ◽  
2001 ◽  
Vol 157 (2) ◽  
pp. 803-815
Author(s):  
Jeffrey J Roix ◽  
Aaron Hagge-Greenberg ◽  
Dennis M Bissonnette ◽  
Sandra Rodick ◽  
Liane B Russell ◽  
...  
Keyword(s):  
Mouse Chromosome ◽  
System Development ◽  
Genomic Region ◽  
Nervous System Development ◽  
Chromosome 14 ◽  
Developmental Defects ◽  
Oak Ridge ◽  
Recessive Mutations ◽  
Large Deletions ◽  
Critical Regions

Abstract The piebald deletion complex is a set of overlapping chromosomal deficiencies surrounding the endothelin receptor B locus collected during the Oak Ridge specific-locus-test mutagenesis screen. These chromosomal deletions represent an important resource for genetic studies to dissect the functional content of a genomic region, and several developmental defects have been associated with mice homozygous for distinct piebald deletion alleles. We have used molecular markers to order the breakpoints for 20 deletion alleles that span a 15.7–18-cM region of distal mouse chromosome 14. Large deletions covering as much as 11 cM have been identified that will be useful for regionally directed mutagenesis screens to reveal recessive mutations that disrupt development. Deletions identified as having breakpoints positioned within previously described critical regions have been used in complementation studies to further define the functional intervals associated with the developmental defects. This has focused our efforts to isolate genes required for newborn respiration and survival, skeletal patterning and morphogenesis, and central nervous system development.

scholarly journals 3D reconstruction of genomic regions from sparse interaction data

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Julen Mendieta-Esteban ◽  
Marco Di Stefano ◽  
David Castillo ◽  
Irene Farabella ◽  
Marc A Marti-Renom
Keyword(s):  
3D Models ◽  
Genomic Region ◽  
Gene Promoters ◽  
Chromosome Conformation ◽  
The Matrix ◽  
Chromatin Structural ◽  
A Cell ◽  
Similar Accuracy ◽  
Genomic Regions ◽  
Interaction Matrices

Abstract Chromosome conformation capture (3C) technologies measure the interaction frequency between pairs of chromatin regions within the nucleus in a cell or a population of cells. Some of these 3C technologies retrieve interactions involving non-contiguous sets of loci, resulting in sparse interaction matrices. One of such 3C technologies is Promoter Capture Hi-C (pcHi-C) that is tailored to probe only interactions involving gene promoters. As such, pcHi-C provides sparse interaction matrices that are suitable to characterize short- and long-range enhancer–promoter interactions. Here, we introduce a new method to reconstruct the chromatin structural (3D) organization from sparse 3C-based datasets such as pcHi-C. Our method allows for data normalization, detection of significant interactions and reconstruction of the full 3D organization of the genomic region despite of the data sparseness. Specifically, it builds, with as low as the 2–3% of the data from the matrix, reliable 3D models of similar accuracy of those based on dense interaction matrices. Furthermore, the method is sensitive enough to detect cell-type-specific 3D organizational features such as the formation of different networks of active gene communities.

scholarly journals Relationship between Yield Components and Partial Resistance to Lecanicillium fungicola in the Button Mushroom, Agaricus bisporus, Assessed by Quantitative Trait Locus Mapping

2012 ◽  
Vol 78 (7) ◽  
pp. 2435-2442 ◽  
Author(s):  
Marie Foulongne-Oriol ◽  
Anne Rodier ◽  
Jean-Michel Savoie
Keyword(s):  
Quantitative Trait ◽  
Wild Strain ◽  
Genomic Region ◽  
Yield Component ◽  
Phenotypic Variance ◽  
Button Mushroom ◽  
Content Type ◽  
Dry Bubble ◽  
Trait Locus ◽  
Genomic Regions

ABSTRACTDry bubble, caused byLecanicillium fungicola, is one of the most detrimental diseases affecting button mushroom cultivation. In a previous study, we demonstrated that breeding for resistance to this pathogen is quite challenging due to its quantitative inheritance. A second-generation hybrid progeny derived from an intervarietal cross between a wild strain and a commercial cultivar was characterized forL. fungicolaresistance under artificial inoculation in three independent experiments. Analysis of quantitative trait loci (QTL) was used to determine the locations, numbers, and effects of genomic regions associated with dry-bubble resistance. Four traits related to resistance were analyzed. Two to four QTL were detected per trait, depending on the experiment. Two genomic regions, on linkage group X (LGX) and LGVIII, were consistently detected in the three experiments. The genomic region on LGX was detected for three of the four variables studied. The total phenotypic variance accounted for by all QTL ranged from 19.3% to 42.1% over all traits in all experiments. For most of the QTL, the favorable allele for resistance came from the wild parent, but for some QTL, the allele that contributed to a higher level of resistance was carried by the cultivar. Comparative mapping with QTL for yield-related traits revealed five colocations between resistance and yield component loci, suggesting that the resistance results from both genetic factors and fitness expression. The consequences for mushroom breeding programs are discussed.

scholarly journals Discovering functional sequences with RELICS, an analysis method for tiling CRISPR screens

2019 ◽  
Author(s):  
Patrick C. Fiaux ◽  
Hsiuyi V. Chen ◽  
Aaron R. Chen ◽  
Poshen B. Chen ◽  
Graham McVicker
Keyword(s):  
New Technology ◽  
Regulatory Sequences ◽  
Analysis Method ◽  
Genome Sequences ◽  
Bayesian Hierarchical ◽  
Guide Rna ◽  
Target Sites ◽  
Cellular Phenotypes ◽  
Genomic Regions ◽  
Crispr Activation

AbstractCRISPR screens are a powerful new technology for the identification of genome sequences that affect cellular phenotypes such as gene expression, survival, and proliferation. By tiling single-guide RNA (sgRNA) target sites across large genomic regions, CRISPR screens have the potential to systematically discovery novel functional sequences, however, a lack of purpose-built analysis tools limits the effectiveness of this approach. Here we describe RELICS, a Bayesian hierarchical model for the discovery of functional sequences from tiling CRISPR screens. RELICS considers the overlapping effects of multiple nearby functional sequences, accounts for the ‘area of effect’ surrounding sgRNA target sites, models overdispersion in sgRNA counts, combines information across multiple pools, and estimates the number of functional sequences supported by the data. In simulations, RELICS outperforms existing methods and provides higher resolution predictions. We apply RELICS to published CRISPR interference and CRISPR activation screens and predict novel regulatory sequences, several of which we experimentally validate. In summary, RELICS is a powerful new analysis method for tiling CRISPR screens that enables the discovery of functional sequences with unprecedented resolution and accuracy.

scholarly journals Regulatory elements in the upstream region of metallothionein gene in tilapia species

2021 ◽  
Vol 30 (1) ◽  
pp. 95-103
Author(s):  
Mohammad Shamimul Alam ◽  
Israt Jahan ◽  
Sadniman Rahman ◽  
Hawa Jahan ◽  
Kaniz Fatema
Keyword(s):  
Tissue Specificity ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Metal Resistance ◽  
Genomic Region ◽  
Upstream Region ◽  
Upstream Sequence ◽  
Oreochromis Aureus ◽  
Metallothionein Gene ◽  
Genomic Regions

Tilapia is a hardy fish which can survive in water bodies polluted with heavy metals. Metal resistance is conferred by higher expression of metallothionein gene (mt) in many organisms. Level, time and tissue-specificity of gene expression is regulated through transcription factor binding sites (TFBS) which may be present in the upstream, downstream, or even in the introns of a gene. So, as a candidate regulatory region, the 5’upstream sequence of mt gene in three tilapia species, Oreochromis aureus, O. niloticus and O. mossambicus was studied. The targeted region was PCR-amplified and then sequenced using a pair of custom-designed primer. A total of only 2.7% variation was found in the sequenced genomic region among the three species. Metal-related TFBS were predicted from these sequences. A total of twenty eight TFBS were found in O. aureus and twenty nine in O. mossambicus and O. niloticus. The number of metalrelated TFBS predicted in the targeted sequence was significantly higher compared to that found in randomly selected other genomic regions of same size from O. niloticus genome. Thus, the results suggest the presence of putative regulatory elements in the targeted upstream region which might have important role in the regulation of mt gene function. Dhaka Univ. J. Biol. Sci. 30(1): 95-103, 2021 (January)

scholarly journals The Caenorhabditis elegans locus lin-15, a negative regulator of a tyrosine kinase signaling pathway, encodes two different proteins.

Genetics ◽  
1994 ◽  
Vol 137 (4) ◽  
pp. 987-997 ◽  
Author(s):  
S G Clark ◽  
X Lu ◽  
H R Horvitz
Keyword(s):  
Caenorhabditis Elegans ◽  
Tyrosine Kinase ◽  
Signaling Pathway ◽  
Genomic Region ◽  
Negative Regulator ◽  
Ras Signaling ◽  
Intercellular Signaling ◽  
Tyrosine Kinase Signaling ◽  
Genomic Regions ◽  
Mrna Precursor

Abstract The Caenorhabditis elegans locus lin-15 negatively regulates an intercellular signaling process that induces formation of the hermaphrodite vulva. The lin-15 locus controls two separate genetic activities. Mutants that lack both activities have multiple, ectopic pseudo-vulvae resulting from the overproduction of vulval cells, whereas mutants defective in only one lin-15 activity appear wild-type. lin-15 acts non-cell-autonomously to prevent the activation of a receptor tyrosine kinase/ras signaling pathway. We report here the molecular characterization of the lin-15 locus. The two lin-15 activities are encoded by contiguous genomic regions and by two distinct, non-overlapping transcripts that may be processed from a single mRNA precursor by trans-splicing. Based on the DNA sequence, the 719- and 1,440-amino acid lin-15 proteins are not similar to each other or to known proteins. lin-15 multivulva mutants, which are defective in both lin-15 activities, contain deletions and insertions that affect the lin-15 genomic region.

Wingless signaling in the Drosophila embryo: zygotic requirements and the role of the frizzled genes

Development ◽  
1999 ◽  
Vol 126 (3) ◽  
pp. 577-586 ◽  
Author(s):  
H. Muller ◽  
R. Samanta ◽  
E. Wieschaus
Keyword(s):  
Genomic Region ◽  
Drosophila Embryo ◽  
Surface Molecules ◽  
New Genes ◽  
Wingless Signaling ◽  
Frizzled Receptors ◽  
Signaling Receptors ◽  
Genomic Regions ◽  
High Degree

Wingless signaling plays a central role during epidermal patterning in Drosophila. We have analyzed zygotic requirements for Wingless signaling in the embryonic ectoderm by generating synthetic deficiencies that uncover more than 99% of the genome. We found no genes required for initial wingless expression, other than previously identified segmentation genes. In contrast, maintenance of wingless expression shows a high degree of zygotic transcriptional requirements. Besides known genes, we have identified at least two additional genomic regions containing new genes involved in Wingless maintenance. We also assayed for the zygotic requirements for Wingless response and found that no single genomic region was required for the cytoplasmic accumulation of Armadillo in the receiving cells. Surprisingly, embryos homozygously deleted for the candidate Wingless receptor, Dfrizzled2, showed a normal Wingless response. However, the Armadillo response to Wingless was strongly reduced in double mutants of both known members of the frizzled family in Drosophila, frizzled and Dfrizzled2. Based on their expression pattern during embryogenesis, different Frizzled receptors may play unique but overlapping roles in development. In particular, we suggest that Frizzled and Dfrizzled2 are both required for Wingless autoregulation, but might be dispensable for late Engrailed maintenance. While Wingless signaling in embryos mutant for frizzled and Dfrizzled2 is affected, Wingless protein is still internalized into cells adjacent to wingless-expressing cells. Incorporation of Wingless protein may therefore involve cell surface molecules in addition to the genetically defined signaling receptors of the frizzled family.

Abstract 2723: Large Genomic Deletions in Plakophilin-2 are a Rare Cause of ARVD/C and ARVD/C-like Disease.

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Jasper J van der Smagt ◽  
Moniek G Cox ◽  
Marcel R Nelen ◽  
J P van Tintelen ◽  
Marc M Entius ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Copy Number ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Task Force ◽  
Direct Sequencing ◽  
Null Alleles ◽  
Loss Of Function ◽  
Genomic Deletions ◽  
Large Deletions ◽  
Plakophilin 2

Introduction: Plakophilin-2 (PKP2) is the major genetic determinant of ARVD/C in the Netherlands. Mutations have been found in 43 out of 82 index cases (52%), diagnosed in accordance with the 1994 Task Force criteria (TFC). A total of 15 different mutations were detected, most of them nonsense (7), frameshift (2) and splice mutations (3), suggesting haploinsufficiency (loss of function) of PKP2 as the predominant mechanism in the etiology of ARVD/C. Since many of these mutations are probably functional null alleles, it was hypothesized that large deletions within - or encompassing - the PKP2 gene can also predispose to ARVD/C. Aim: to determine whether large deletions in PKP2 underlie apparently PKP2 negative ARVD/C cases. Methods: a multiple ligation-dependent probe amplification kit was developed to determine copy number of all 14 exons of the PKP2 gene. In total 120 patients were included that had been referred for PKP2 testing, and that had tested negative with direct sequencing or denaturing gradient gel electrophoresis. Twenty-six patients had definite ARVD/C (TFC: ≥ 4 pts), 11 patients scored 3 pts and 83 patients scored less than 3 pts or had insufficient clinical data. Results: a single deletion was detected encompassing the first 4 exons and promoter region of the PKP2 gene. The patient, a 35 year old male, had experienced near-syncope during exercise. Mild dilation of the right ventricle (1 pt), negative T-waves in V1-V5 (1 pt) and short VT’s with LBBB morphology during exercise testing were found (1 pt). There were frequent PVC’s. The father also carried the deletion. A third degree paternal relative died suddenly at age 28 during exercise. Conclusion: large genomic deletions of PKP2, that go unnoticed with sequencing, can cause ARVD/C-like disease. Comprehensive testing of PKP2 should include assessment of copy number. However, the frequency of large deletions seems to be low. Since the detected deletion includes the promoter and the transcription initiation site, no protein can be derived from the affected allele. This patient demonstrates that complete loss of one PKP2 allele does not necessarily lead to more severe ARVD/C. This is in keeping with the concept of haploinsufficiency and the assumption that many PKP2 mutations cause functional null alleles.

Basic principles of genetic disease

ESC CardioMed ◽  
2018 ◽  
pp. 669-671
Author(s):  
Eric Schulze-Bahr
Keyword(s):  
Genetic Disease ◽  
Copy Number ◽  
Copy Number Variants ◽  
Single Nucleotide Variants ◽  
Individual Genome ◽  
Base Pairs ◽  
Single Nucleotide ◽  
Basic Principles ◽  
Bona Fide ◽  
Genomic Regions

The human genome consists of approximately 3 billion (3 × 109) base pairs of DNA (around 20,000 genes), organized as 23 chromosomes (diploid parental set), and a small mitochondrial genome (37 genes, including 13 proteins; 16,589 base pairs) of maternal origin. Most human genetic variation is natural, that is, common or rare (minor allele frequency >0.1%) and does not cause disease—apart from every true disease-causing (bona fide) mutation each individual genome harbours more than 3.5 million single nucleotide variants (including >10,000 non-synonymous changes causing amino acid substitutions) and 200–300 large structural or copy number variants (insertions/deletions, up to several thousands of base-pairs) that are non-disease-causing variations and scattered throughout coding and non-coding genomic regions.

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