Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line
Autor: | E. N. Antonova, Pavel Volchkov, Natalya Grebenkina, N. A. Volkova, Aykaz Eremyan, Svetlana Zvereva, Anna Gaponova, Olga Glazova |
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Jazyk: | angličtina |
Rok vydání: | 2018 |
Předmět: |
0301 basic medicine
endogenous promoter tandem expression Locus (genetics) homology directed repair Biology General Biochemistry Genetics and Molecular Biology Homology directed repair 03 medical and health sciences 0302 clinical medicine Genome editing CRISPR General Pharmacology Toxicology and Pharmaceutics Gene CRISPR/Cas9 targeting General Immunology and Microbiology Cas9 chicken DF-1 cell line General Medicine Articles Cell biology 030104 developmental biology GAPDH Gene Homologous recombination 030217 neurology & neurosurgery Research Article |
Zdroj: | F1000Research |
ISSN: | 2046-1402 |
Popis: | 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. |
Databáze: | OpenAIRE |
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