scholarly journals The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Swati Jivanji ◽  
Chad Harland ◽  
Sally Cole ◽  
Brigid Brophy ◽  
Dorian Garrick ◽  
...  
Keyword(s):  
Cell Lines ◽  
Gene Editing ◽  
Agricultural Sector ◽  
De Novo ◽  
Animal Health ◽  
Coat Colour ◽  
Structural Variations ◽  
Depth Analysis ◽  
Breeding Schemes ◽  
Selection Of

Abstract Background Animal health and welfare are at the forefront of public concern and the agricultural sector is responding by prioritising the selection of welfare-relevant traits in their breeding schemes. In some cases, welfare-enhancing traits such as horn-status (i.e., polled) or diluted coat colour, which could enhance heat tolerance, may not segregate in breeds of primary interest, highlighting gene-editing tools such as the CRISPR-Cas9 technology as an approach to rapidly introduce variation into these populations. A major limitation preventing the acceptance of CRISPR-Cas9 mediated gene-editing, however, is the potential for off-target mutagenesis, which has raised concerns about the safety and ultimate applicability of this technology. Here, we present a clone-based study design that has allowed a detailed investigation of off-target and de novo mutagenesis in a cattle line bearing edits in the PMEL gene for diluted coat-colour. Results No off-target events were detected from high depth whole genome sequencing performed in precursor cell-lines and resultant calves cloned from those edited and non-edited cell lines. Long molecule sequencing at the edited site and plasmid-specific PCRs did not reveal structural variations and/or plasmid integration events in edited samples. Furthermore, an in-depth analysis of de novo mutations across the edited and non-edited cloned calves revealed that the mutation frequency and spectra were unaffected by editing status. Cells in culture, however, appeared to have a distinct mutation signature where de novo mutations were predominantly C > A mutations, and in cloned calves they were predominantly T > G mutations, deviating from the expected excess of C > T mutations. Conclusions We found no detectable CRISPR-Cas9 associated off-target mutations in the gene-edited cells or calves derived from the gene-edited cell line. Comparison of de novo mutation in two gene-edited calves and three non-edited control calves did not reveal a higher mutation load in any one group, gene-edited or control, beyond those anticipated from spontaneous mutagenesis. Cell culture and somatic cell nuclear transfer cloning processes contributed the major source of contrast in mutational profile between samples.

scholarly journals The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis

2021 ◽  
Author(s):  
Swati Jivanji ◽  
Chad Harland ◽  
Sally Cole ◽  
Brigid Brophy ◽  
Dorian Garrick ◽  
...  
Keyword(s):  
Cell Lines ◽  
Gene Editing ◽  
Agricultural Sector ◽  
De Novo ◽  
Animal Health ◽  
Coat Colour ◽  
Structural Variations ◽  
Depth Analysis ◽  
Breeding Schemes ◽  
Selection Of

AbstractAnimal health and welfare are at the forefront of public concern and the agricultural sector is responding by prioritising the selection of welfare-relevant traits in their breeding schemes. In some cases, welfare-enhancing traits such as horn-status (i.e., polled) or diluted coat colour, which could enhance heat tolerance, may not segregate in breeds of primary interest, highlighting gene-editing tools such as the CRISPR-Cas9 technology as an approach to rapidly introduce variation into these populations. A major limitation preventing the acceptance of CRISPR-Cas9 mediated gene-editing, however, is the potential for off-target mutagenesis, which has raised concerns about the safety and ultimate applicability of this technology. Here, we present a clone-based study design that has allowed a detailed investigation of off-target and de novo mutagenesis in a cattle line bearing edits in the PMEL gene for diluted coat-colour. No off-target events were detected from high depth whole genome sequencing performed in precursor cell-lines and resultant calves cloned from those edited and non-edited cell lines. Long molecule sequencing at the edited site and plasmid-specific PCRs did not reveal structural variations and/or plasmid integration events in edited samples. Furthermore, an in-depth analysis of de novo mutations across samples revealed that the mutation frequency and spectra were unaffected by editing status. Cells in culture, however, had a distinct mutation signature where de novo mutations were predominantly C>A mutations, and in cloned calves they were predominantly T>G mutations, deviating from the expected excess of C>T mutations. We conclude that the gene-edited cells and calves in this study did not present a higher mutation load than unedited controls. Cell culture and somatic cell nuclear transfer cloning processes contributed the major source of contrast in mutational profile between samples.

Increasing gene editing efficiencies in eukaryotic cell lines by selection of appropriate CRISPR-Cas9 reagents

Cytotherapy ◽  
2015 ◽  
Vol 17 (6) ◽  
pp. S53
Author(s):  
Melissa L. Kelley ◽  
Zaklina Strezoska ◽  
Hidevaldo Machado ◽  
Emily Anderson ◽  
Maren Mayer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Gene Editing ◽  
Eukaryotic Cell ◽  
Selection Of

Posttransplant CMV infection and the role of immunosuppression (Sponsor: Novartis Pharma GmbH, Nürnberg)

2016 ◽  
Vol 04 (01) ◽  
pp. 4-10
Keyword(s):  
Lower Incidence ◽  
Paradigm Shift ◽  
Mtor Inhibitor ◽  
De Novo ◽  
Expert Panel ◽  
Risk Of Infection ◽  
Cmv Infection ◽  
Expert Meeting ◽  
Selection Of

AbstractImmunosuppression permits graft survival after transplantation and consequently a longer and better life. On the other hand, it increases the risk of infection, for instance with cytomegalovirus (CMV). However, the various available immunosuppressive therapies differ in this regard. One of the first clinical trials using de novo everolimus after kidney transplantation [1] already revealed a considerably lower incidence of CMV infection in the everolimus arms than in the mycophenolate mofetil (MMF) arm. This result was repeatedly confirmed in later studies [2–4]. Everolimus is now considered a substance with antiviral properties. This article is based on the expert meeting “Posttransplant CMV infection and the role of immunosuppression”. The expert panel called for a paradigm shift: In a CMV prevention strategy the targeted selection of the immunosuppressive therapy is also a key element. For patients with elevated risk of CMV, mTOR inhibitor-based immunosuppression is advantageous as it is associated with a significantly lower incidence of CMV events.

Tobacco’s Appeal to the Senses and the Early Modern Smoker’s Still Life

2020 ◽  
pp. 123-150
Author(s):  
Julia Saviello
Keyword(s):  
Still Life ◽  
Sense Data ◽  
Complex Image ◽  
Starting Point ◽  
Depth Analysis ◽  
Tobacco Pipes ◽  
Five Senses ◽  
The Senses ◽  
Cut Tobacco ◽  
Selection Of

Smell and taste – of the five senses these are the two most strongly stimulated by smoking tobacco. The article presents an in-depth analysis of the reflection of both these forms of sensory perception in textual and visual sources concerning the early consumption of the herb. In a first step, tobacco’s changing reception, first as medicine and then as stimulant, is traced through the years of its increasing distribution in Europe, starting in the middle of the 16th century. As this overview reveals, at that time the still little known substance gave rise to new forms of sense perception. Following recent studies on smell and gustation, which have stressed the need to take into account the interactions between these senses, the article probes the manifold stimulation of the senses by tobacco with reference to allegorical representations and genre scenes addressing the five senses. The smoking of tobacco was thematized in both of these art forms as a means of visualizing either smell or taste. Yet, these depictions show no indication of any deliberate engagement with the exchange of sense data between mouth and nose. The question posed at the end of this paper is whether this holds true also for early smoker’s still lifes. In the so-called toebakjes or rookertjes, a subgenre of stilllife painting that, like tobacco, was still a novelty at the beginning of the 17th century, various smoking paraphernalia – such as rolled or cut tobacco, pipes and tins – are arrayed with various kinds of foods and drinks. Finally, the article addresses a selection of such smoker’s still lifes, using the toebakje by Pieter Claesz., probably the first of its kind, as a starting point and the work by Georg Flegel as a comparative example. Through their selection of objects, both offer a complex image of how tobacco engages different senses.

scholarly journals A study of transposable element-associated structural variations (TASVs) using a de novo-assembled Korean genome

2021 ◽  
Author(s):  
Seyoung Mun ◽  
Songmi Kim ◽  
Wooseok Lee ◽  
Keunsoo Kang ◽  
Thomas J. Meyer ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Genome Assembly ◽  
De Novo ◽  
Personal Genome ◽  
Human Populations ◽  
Whole Genome ◽  
Structural Variations ◽  
Insert Size ◽  
Human Genomes ◽  
Next Generation Sequencing Ngs

AbstractAdvances in next-generation sequencing (NGS) technology have made personal genome sequencing possible, and indeed, many individual human genomes have now been sequenced. Comparisons of these individual genomes have revealed substantial genomic differences between human populations as well as between individuals from closely related ethnic groups. Transposable elements (TEs) are known to be one of the major sources of these variations and act through various mechanisms, including de novo insertion, insertion-mediated deletion, and TE–TE recombination-mediated deletion. In this study, we carried out de novo whole-genome sequencing of one Korean individual (KPGP9) via multiple insert-size libraries. The de novo whole-genome assembly resulted in 31,305 scaffolds with a scaffold N50 size of 13.23 Mb. Furthermore, through computational data analysis and experimental verification, we revealed that 182 TE-associated structural variation (TASV) insertions and 89 TASV deletions contributed 64,232 bp in sequence gain and 82,772 bp in sequence loss, respectively, in the KPGP9 genome relative to the hg19 reference genome. We also verified structural differences associated with TASVs by comparative analysis with TASVs in recent genomes (AK1 and TCGA genomes) and reported their details. Here, we constructed a new Korean de novo whole-genome assembly and provide the first study, to our knowledge, focused on the identification of TASVs in an individual Korean genome. Our findings again highlight the role of TEs as a major driver of structural variations in human individual genomes.

scholarly journals The ‘3CE2CE’ Framework—Change Management Towards a Circular Economy: Opportunities for Agribusiness

2021 ◽  
Author(s):  
Nan-Hua Nadja Yang ◽  
Ana Carolina Bertassini ◽  
Jéssica Alves Justo Mendes ◽  
Mateus Cecílio Gerolamo
Keyword(s):  
Content Analysis ◽  
Literature Review ◽  
Change Management ◽  
Circular Economy ◽  
Agricultural Sector ◽  
Implementation Process ◽  
Managerial Practices ◽  
Depth Analysis ◽  
The Common ◽  
Transformation Processes

AbstractFor the transition towards a circular economy (CE), organisations have to be prepared to adapt to major changes. Thus, the concept and implementation of change management (CM) will be essential to an organisation’s success during this transformative period. Studies have shown that organisational CE barriers were more significant than individual CE barriers. To overcome such obstacles, the most appropriate set of managerial practices should be carefully considered. These barriers also have the potential to influence the agricultural sector, which seeks to adopt more sustainable ways of production. The goal of this paper is to propose a solution framework based on CM strategies to overcome organisational challenges posed by a CE, especially for agribusinesses. To accomplish this objectively, a systematic literature review and a content analysis were conducted. The common errors in CM within the implementation process and the main CE barriers were identified and classified. An in-depth analysis of the issue’s roots led to a solid understanding of how to tackle such CM problems. This paper presents an overview of organisational CE barriers verified in the agricultural context, the common errors in CM, and the correlation between these findings. The two areas were then combined in a matrix that shows the connection between common errors in CM errors and CE barriers. Based on this result, a solution framework called 3CE2CE was developed that provides a step-by-step guide on how organisations can successfully undergo transformation processes towards a CE with the principles of CM.

scholarly journals Examination of the Transcriptional Response to LaMIR166a Overexpression in Larix kaempferi (Lamb.) Carr

Biology ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 576
Author(s):  
Yanru Fan ◽  
Wanfeng Li ◽  
Zhexin Li ◽  
Shaofei Dang ◽  
Suying Han ◽  
...  
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
De Novo ◽  
Pearson Correlation ◽  
Transcriptional Response ◽  
Correlation Coefficients ◽  
Embryonic Cell ◽  
Larix Kaempferi ◽  
Transgenic Cell

The study of somatic embryogenesis can provide insight into early plant development. We previously obtained LaMIR166a-overexpressing embryonic cell lines of Larix kaempferi (Lamb.) Carr. To further elucidate the molecular mechanisms associated with miR166 in this species, the transcriptional profiles of wild-type (WT) and three LaMIR166a-overexpressing transgenic cell lines were subjected to RNA sequencing using the Illumina NovaSeq 6000 system. In total, 203,256 unigenes were generated using Trinity de novo assembly, and 2467 differentially expressed genes were obtained by comparing transgenic and WT lines. In addition, we analyzed the cleaved degree of LaMIR166a target genes LaHDZ31–34 in different transgenic cell lines by detecting the expression pattern of LaHdZ31–34, and their cleaved degree in transgenic cell lines was higher than that in WT. The downstream genes of LaHDZ31–34 were identified using Pearson correlation coefficients. Yeast one-hybrid and dual-luciferase report assays revealed that the transcription factors LaHDZ31–34 could bind to the promoters of LaPAP, LaPP1, LaZFP5, and LaPHO1. This is the first report of gene expression changes caused by LaMIR166a overexpression in Japanese larch. These findings lay a foundation for future studies on the regulatory mechanism of miR166.

scholarly journals A long reads-based de-novo assembly of the genome of the Arlee homozygous line reveals chromosomal rearrangements in rainbow trout

2021 ◽  
Author(s):  
Guangtu Gao ◽  
Susana Magadan ◽  
Geoffrey C Waldbieser ◽  
Ramey C Youngblood ◽  
Paul A Wheeler ◽  
...  
Keyword(s):  
Rainbow Trout ◽  
Chromosome Number ◽  
Genome Assembly ◽  
De Novo Assembly ◽  
De Novo ◽  
Sequence Data ◽  
Structural Variations ◽  
High Coverage ◽  
Haploid Chromosome Number ◽  
Long Reads

Abstract Currently, there is still a need to improve the contiguity of the rainbow trout reference genome and to use multiple genetic backgrounds that will represent the genetic diversity of this species. The Arlee doubled haploid line was originated from a domesticated hatchery strain that was originally collected from the northern California coast. The Canu pipeline was used to generate the Arlee line genome de-novo assembly from high coverage PacBio long-reads sequence data. The assembly was further improved with Bionano optical maps and Hi-C proximity ligation sequence data to generate 32 major scaffolds corresponding to the karyotype of the Arlee line (2 N = 64). It is composed of 938 scaffolds with N50 of 39.16 Mb and a total length of 2.33 Gb, of which ∼95% was in 32 chromosome sequences with only 438 gaps between contigs and scaffolds. In rainbow trout the haploid chromosome number can vary from 29 to 32. In the Arlee karyotype the haploid chromosome number is 32 because chromosomes Omy04, 14 and 25 are divided into six acrocentric chromosomes. Additional structural variations that were identified in the Arlee genome included the major inversions on chromosomes Omy05 and Omy20 and additional 15 smaller inversions that will require further validation. This is also the first rainbow trout genome assembly that includes a scaffold with the sex-determination gene (sdY) in the chromosome Y sequence. The utility of this genome assembly is demonstrated through the improved annotation of the duplicated genome loci that harbor the IGH genes on chromosomes Omy12 and Omy13.

Cybersecurity Standards in the Context of Operating System

2021 ◽  
Vol 54 (3) ◽  
pp. 1-36
Author(s):  
Syed Wasif Abbas Hamdani ◽  
Haider Abbas ◽  
Abdul Rehman Janjua ◽  
Waleed Bin Shahid ◽  
Muhammad Faisal Amjad ◽  
...  
Keyword(s):  
Organizational Context ◽  
Cyber Attacks ◽  
Fine Tuning ◽  
Security Vulnerabilities ◽  
Compliance Testing ◽  
Depth Analysis ◽  
Minimum Requirements ◽  
Security Standards ◽  
High Degree ◽  
Selection Of

Cyber threats have been growing tremendously in recent years. There are significant advancements in the threat space that have led towards an essential need for the strengthening of digital infrastructure security. Better security can be achieved by fine-tuning system parameters to the best and optimized security levels. For the protection of infrastructure and information systems, several guidelines have been provided by well-known organizations in the form of cybersecurity standards. Since security vulnerabilities incur a very high degree of financial, reputational, informational, and organizational security compromise, it is imperative that a baseline for standard compliance be established. The selection of security standards and extracting requirements from those standards in an organizational context is a tedious task. This article presents a detailed literature review, a comprehensive analysis of various cybersecurity standards, and statistics of cyber-attacks related to operating systems (OS). In addition to that, an explicit comparison between the frameworks, tools, and software available for OS compliance testing is provided. An in-depth analysis of the most common software solutions ensuring compliance with certain cybersecurity standards is also presented. Finally, based on the cybersecurity standards under consideration, a comprehensive set of minimum requirements is proposed for OS hardening and a few open research challenges are discussed.

scholarly journals Germline PRKACA amplification causes variable phenotypes that may depend on the extent of the genomic defect: molecular mechanisms and clinical presentations

2015 ◽  
Vol 172 (6) ◽  
pp. 803-811 ◽  
Author(s):  
Maya B Lodish ◽  
Bo Yuan ◽  
Isaac Levy ◽  
Glenn D Braunstein ◽  
Charalampos Lyssikatos ◽  
...  
Keyword(s):  
Copy Number ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Genetic Defect ◽  
Molecular Cytogenetics ◽  
Case Series ◽  
Sporadic Case ◽  
Genomic Rearrangements ◽  
Clinical Presentations ◽  
Depth Analysis

ObjectiveWe have recently reported five patients with bilateral adrenocortical hyperplasia (BAH) and Cushing's syndrome (CS) caused by constitutive activation of the catalytic subunit of protein kinase A (PRKACA). By doing new in-depth analysis of their cytogenetic abnormality, we attempted a better genotype–phenotype correlation of theirPRKACAamplification.DesignThis study is a case series.MethodsMolecular cytogenetic, genomic, clinical, and histopathological analyses were performed in five patients with CS.ResultsReinvestigation of the defects of previously described patients by state-of-the-art molecular cytogenetics showed complex genomic rearrangements in the chromosome 19p13.2p13.12 locus, resulting in copy number gains encompassing the entirePRKACAgene; three patients (one sporadic case and two related cases) were observed with gains consistent with duplications, while two sporadic patients were observed with gains consistent with triplications. Although all five patients presented with ACTH-independent CS, the three sporadic patients had micronodular BAH and underwent bilateral adrenalectomy in early childhood, whereas the two related patients, a mother and a son, presented with macronodular BAH as adults. In at least one patient,PRKACAtriplication was associated with a more severe phenotype.ConclusionsConstitutional chromosomalPRKACAgene amplification is a recently identified genetic defect associated with CS, a trait that may be inherited in an autosomal dominant manner or occurde novo. Genomic rearrangements can be complex and can result in different copy number states of dosage-sensitive genes, e.g., duplication and triplication.PRKACAamplification can lead to variable phenotypes clinically and pathologically, both micro- and macro-nodular BAH, the latter of which we speculate may depend on the extent of amplification.

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