Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system

Bevacqua, R. J.; Fernández-Martı́n, Rafael; Savy, Virginia; Canel, N. G.; Gismondi, María Inés; Kues, Wilfried A.; Carlson, Daniel F.; Fahrenkrug, Scott C.; Niemann, Heiner; Taboga, Oscar; Ferraris, S.; Salamone, D.

doi:10.1016/j.theriogenology.2016.06.010

Cited by 70 publications

(36 citation statements)

References 68 publications

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“…Prion diseases represented a convenient experimental ground for testing the PPI-FIT approach, as compelling evidence indicates that prion replication and toxicity can be inhibited by targeting a single protein (PrP) 24,25,44,45 . Theoretical options to achieve this goal include the ablation of the PrP gene, for example by using the CRISPR technology 79,80 , decreasing its synthesis with specific antisense oligonucleotides (ASOs) 27 , or acting directly on cell surface PrP 81,82 , for example with ligands stabilizing the native fold and inhibiting its conversion into PrP Sc 50,77 . Despite encouraging results obtained with ASOs recently, so far, none of these approaches has been translated into effective therapies for prion diseases.…”

Section: Discussionmentioning

confidence: 99%

Pharmacological Protein Inactivation by Targeting Folding Intermediates

Spagnolli

Massignan

Astolfi

et al. 2020

Preprint

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Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. Using these techniques to study the negative regulation of the androgen receptor (AR), we discovered a key functional role played by non-native metastable states appearing along the folding pathway of this protein. This unexpected observation inspired us to design a completely novel drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein expression by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP, and identified four different small molecule ligands for this conformer, all capable of selectively lowering the expression of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression. INTRODUCTIONProtein expression and function in eukaryotic cells are tightly harmonized processes modulated by the combination of different layers of regulation. These may occur at the level of gene transcription, processing, stability, and translation of the mRNA as well as by assembly, post-translational modifications, sorting, recycling, and degradation of the corresponding polypeptide 1 . In addition, the expression of a small subset of proteins is known to be regulated by the presence of specific ligands, which are required along the folding pathway to reach the native, functional state 2-7 . For example, the folding of the kinase-inducible domain of the cAMP-response-element-binding protein is known to proceed only upon interaction with the CREB-binding protein 8 . Other typical examples of liganddependent folding mechanism include nuclear receptors, such as estrogen and androgen receptors 9 . The integration between these pathways and the protein quality control machinery, deputed to avoid the production and accumulation of aberrantly folded proteins, is known as proteostasis 1,10 . Mechanisms by which proteostasis is ensured include chaperone-assisted protein folding as well as re-routing and degradation of misfolded protein conformers. These processes play a fundamental role during development, aging, tolerance to environmental stresses, and minimize alterations of proteome homeostasis induced by pathogens 11 . Consistently, a large percentage of human pathologies are linked to alterations of proteostasis, including a broad spectrum of age-related brain disorders, known as neurodegenerative diseases, ...

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Section: Discussionmentioning

confidence: 99%

Pharmacological Protein Inactivation by Targeting Folding Intermediates

Spagnolli

Massignan

Astolfi

et al. 2020

Preprint

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“…Polled locus is responsible for the growth of horn in cattle and using TALEN this locus was knocked out in Holstein cattle to produce hornless animal (Carlson et al, 2016). Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos were done using TALEN (Choi et al, 2015) and CRISPR/Cas9 system (Bevacqua et al, 2016). This strategy can be further used to produce prionfree cattle.…”

Section: Application Of Genome Editors In Livestock Speciesmentioning

confidence: 99%

Genome Editing by Programmable Nucleases and their applications in livestock species

Zafar¹,

Singh²,

Kumar³

2019

Journal of Livestock Science

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Genetic modification in livestock species is almost 30 years old but many technical issues limited its use. The earlier methods -pronuclear injection and viral integration had several problems including random integration, silencing, low efficiency, inefficient gene expression regulation etc. With the advent of programmable nucleases such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9), precision genome editing has now become possible. Genome editing by these nucleases is an efficient and powerful technique to alter the genome of organisms for site-specific modification and integration of endogenous and exogenous genes respectively. These techniques have already been used in species like Drosophila, zebrafish, mice, rats, monkeys, humans, pigs, cattle, sheep, goats and others. Generation of genome edited livestock has been proved feasible and valuable and they are useful for the understanding of complex physiology, generation of animals with improved traits and disease resistance, producing transgenic animals for the therapeutic proteins and large animal models for human diseases. Given the high efficiency of these techniques, it is expected that a large number of genome edited livestock will be produced in the future. In this review, we provide an overview of the programmable nucleases, their underlying mechanism, and usefulness of genome editing in livestock species.

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“…An alternative to the above-described approach for inducing mutations in preimplantation embryos is the injection of target specific nucleases into the zygote. This can induce mutations at a relatively high rate, but the type of mutation is unknown during development of the embryo and it also often results in mosaic mutations [39][40][41][42], hampering analysis and repeatability of experiments. In addition, the limited genomic material per specimen impedes in-depth investigations, especially when combined with imaging procedures or transcriptome analyses.…”

Section: Domestic Species As Model Organisms For Preimplantation Devementioning

confidence: 99%

Comparative aspects of early lineage specification events in mammalian embryos – insights from reverse genetics studies

Simmet¹,

Zakhartchenko²,

Wolf

2018

Cell Cycle

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Within the mammalian class, formation of the blastocyst is morphologically highly conserved among different species. The molecular and cellular events during preimplantation embryo development have been studied extensively in the mouse as model organism, because multiple genetically defined strains and a plethora of reverse genetics tools are available to dissect specific gene functions and regulatory networks. However, major differences in preimplantation developmental kinetics, implantation, and placentation exist among mammalians, and recent studies in species other than mouse showed, that even regulatory mechanisms of the first lineage differentiation events and maintenance of pluripotency are not always conserved. Here, we focus on the first and the second lineage segregation in mouse and bovine embryos, when the first differentiated cell types emerge. We outline their common features and differences in the regulation of these essential events during embryonic development with a glance at further species. In addition, we show how new reverse genetics strategies aid the study of regulatory circuits in embryos of domestic species, enhancing our overall understanding of mammalian preimplantation development.

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Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system

Cited by 70 publications

References 68 publications

Pharmacological Protein Inactivation by Targeting Folding Intermediates

Pharmacological Protein Inactivation by Targeting Folding Intermediates

Genome Editing by Programmable Nucleases and their applications in livestock species

Comparative aspects of early lineage specification events in mammalian embryos – insights from reverse genetics studies

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