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,  том 7 ,  страницы 238

Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line

Ekaterina Antonova 1, 2
, 
Natalya Volkova 3
, 
Olga Glazova 2
, 
Anna Gaponova 1, 2
, 
Aykaz Eremyan 1, 2
, 
Svetlana Zvereva 1, 2
, 
Natalya Grebenkina 1, 2
, 
Natalia B. Volkova 4
, 
Pavel Volchkov 1, 2
1
 
2
 
3
 
Ernst Institute of Animal Husbandry, Podolsk Municipal District, Moscow Region, 142132 , Russian Federation. | 
4
 
Ernst Institute of Animal Husbandry, Podolsk Municipal District, Moscow Region, 142132
Тип публикации: Journal Article
Дата публикации: 2018-05-30
F1000 Research
scimago Q1
БС1
SJR: 0.537
CiteScore: 2.9
Impact factor: —
ISSN: 20461402
General Biochemistry, Genetics and Molecular Biology
General Medicine
General Immunology and Microbiology
General Pharmacology, Toxicology and Pharmaceutics
Краткое описание
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.
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Antonova E. et al. Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line // F1000Research. 2018. Vol. 7. p. 238.
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Antonova E., Volkova N., Glazova O., Gaponova A., Eremyan A., Zvereva S., Grebenkina N., Volkova N. B., Volchkov P. Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line // F1000Research. 2018. Vol. 7. p. 238.
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TY - JOUR
DO - 10.12688/f1000research.13457.2
UR - https://f1000research.com/articles/7-238/v2
TI - Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line
T2 - F1000Research
AU - Antonova, Ekaterina
AU - Volkova, Natalya
AU - Glazova, Olga
AU - Gaponova, Anna
AU - Eremyan, Aykaz
AU - Zvereva, Svetlana
AU - Grebenkina, Natalya
AU - Volkova, Natalia B.
AU - Volchkov, Pavel
PY - 2018
DA - 2018/05/30
PB - F1000 Research
SP - 238
VL - 7
PMID - 29946437
SN - 2046-1402
ER -
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@article{2018_Antonova,
author = {Ekaterina Antonova and Natalya Volkova and Olga Glazova and Anna Gaponova and Aykaz Eremyan and Svetlana Zvereva and Natalya Grebenkina and Natalia B. Volkova and Pavel Volchkov},
title = {Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line},
journal = {F1000Research},
year = {2018},
volume = {7},
publisher = {F1000 Research},
month = {may},
url = {https://f1000research.com/articles/7-238/v2},
pages = {238},
doi = {10.12688/f1000research.13457.2}
}