Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken 3’-untranslated region of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene ( neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9 system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.
There are several technologies for plant genome editing, of which the most simple and universal is CRISPR/ Cas. Currently, this technology is widely used for gene knockout, deleting genome fragments and inserting exogenous sequences in the plant genome. For each of these applications, many different types of genetic tools have been developed that are used by various research groups to solve specific problems. The CRISPR/Cas technology for plant genome editing is at an early stage of optimization, which is reflected by the ongoing search for the most effective, simple and flexible techniques. As a result, experimental work has to be preceded by a rather long and laborious process of selecting a genetic tool that will be optimal for a specific experimental task. In our review we describe the main variants of the CRISPR/Cas technology used to edit a plant genome. We classify them in terms of experimental tasks solved, major components and technology performance. In the first half of the review a detailed description of two major components of CRISPR/Cas technology -nuclease and guide RNAis given, the effect of structural features of these elements on editing efficiency is analyzed. Experimental data on the relationship between editing efficiency and nucleotide sequence of guide RNA are generalized. We also give the characteristic for different variants of nucleases used for plant genome editing and discuss their benefits for different experimental purposes. In the second half of the review various strategies for expression of CRISPR/Cas elements in plant cells, in particular, advantages and disadvantages of stable transformation and transient expression, are discussed. The effect of various regulatory elements of genes encoding nuclease and guide RNA on editing efficiency is described. Special emphasis is placed on the techniques of increasing targeted gene replacement efficiency.Key words: CRISPR/Cas; genome editing; plant genetic engineering; sgRNA.Существует несколько технологий редактирования генома рас-тений, из которых наиболее простой и универсальной является CRISPR/Cas. В настоящее время эта технология активно использу-ется для нокаутирования генов, делеций участков генома и встра-ивания экзогенных последовательностей в растительный геном. Для каждого из этих приложений разработано множество вариан-тов генетического инструментария, которые использовались раз-личными исследовательскими группами для решения конкретных задач. Технология CRISPR/Cas применительно к редактированию генома растений находится на начальном этапе оптимизации, вы-ражающейся в поиске наиболее эффективных, простых и универ-сальных методик. Вследствие этого экспериментальная работа должна предваряться достаточно длительным и трудоемким вы -бором варианта генетического инструментария, оптимального для решения конкретной экспериментальной задачи. В данном обзо-ре мы охарактеризовали разработанные на сегодняшний день основные варианты генетического инструментария технологии CRISPR/ Cas для редактирования генома растений с точки зрения решаем...
CRISPR/Cas9 system is becoming the dominant genome editing Background: tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific.Three gRNAs were chosen to target chicken 3'-untranslated region Methods: of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene ( ) under cytomegalovirus promoter. Correct integration of the neoR cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS).We have established a CRISPR/Cas9 system to target an Results: endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter.The approach can be used further to insert genes of interest Conclusions: under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.
Influenza virus can cause both seasonal infections and unpredictable pandemics. Rapidly evolving avian H5N1 virus is getting increasingly infective for humans. Since avian Influenza can be transmitted by domestic birds, serving as a key link between wild aquatic birds and humans, an effective measure to control the influenza transmission would be eradication of the infection in poultry. It is known that the virus penetrates into the cell through binding with the terminal oligosaccharides - sialic acids (SA) - on the cell surfaces. Removal of SA might be a potential antiviral strategy. An approach to developing chicken lines that are resistant to influenza viruses could be the creation of genetically modified birds. Thus it is necessary to select a gene that provides defense to influenza. Here we have expressed in cells a range of exogenous sialidases and estimated their activity and specificity towards SA residues. Several bacterial, viral and human sialidases were tested. We adopted bacterial sialidases from and for expression on the cell surface by fusing catalytic domains with transmembrane domains. We also selected Influenza A/PuertoRico/8/34/H1N1 neuraminidase and human membrane sialidase () genes. Lectin binding assay was used for estimation of a α (2,3)-sialylation level by fluorescent microscopy and FACS. We compared sialidases from bacteria, Influenza virus and human. Sialidases from and Influenza A neuraminidase effectively cleaved α (2-3)-SA receptors. Viral neuraminidase demonstrated a higher activity. Sialidases from and did not show any activity against α (2-3) SA under physiological conditions. : Our results demonstrated that sialidases with different specificity and activity can be selected as genes providing antiviral defence. Combining chosen sialidases with different activity together with tissue-specific promoters would provide an optimal level of desialilation to prevent infection. Tissue specific expression of the sialidases could protect domestic birds from infection.
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