Quantitative analysis of CRISPR/Cas9-mediated provirus deletion in blue egg layer chicken PGCs by digital PCR
Abstract Primordial germ cells (PGCs), the precursors of sperm and oocytes, pass on the genetic material to the next generation. The previously established culture system of chicken PGCs holds many possibilities for functional genomics studies and the rapid introduction of desired traits. Here, we e...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-09-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-19861-7 |
| _version_ | 1797995463045021696 |
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| author | Stefanie Altgilbers Claudia Dierks Sabine Klein Steffen Weigend Wilfried A. Kues |
| author_facet | Stefanie Altgilbers Claudia Dierks Sabine Klein Steffen Weigend Wilfried A. Kues |
| author_sort | Stefanie Altgilbers |
| collection | DOAJ |
| description | Abstract Primordial germ cells (PGCs), the precursors of sperm and oocytes, pass on the genetic material to the next generation. The previously established culture system of chicken PGCs holds many possibilities for functional genomics studies and the rapid introduction of desired traits. Here, we established a CRISPR/Cas9-mediated genome editing protocol for the genetic modification of PGCs derived from chickens with blue eggshell color. The sequence targeted in the present report is a provirus (EAV-HP) insertion in the 5’-flanking region of the SLCO1B3 gene on chromosome 1 in Araucana chickens, which is supposedly responsible for the blue eggshell color. We designed pairs of guide RNAs (gRNAs) targeting the entire 4.2 kb provirus region. Following transfection of PGCs with the gRNA, genomic DNA was isolated and analyzed by mismatch cleavage assay (T7EI). For absolute quantification of the targeting efficiencies in homozygous blue-allele bearing PGCs a digital PCR was established, which revealed deletion efficiencies of 29% when the wildtype Cas9 was used, and 69% when a high-fidelity Cas9 variant was employed. Subsequent single cell dilutions of edited PGCs yielded 14 cell clones with homozygous deletion of the provirus. A digital PCR assay proved the complete absence of this provirus in cell clones. Thus, we demonstrated the high efficiency of the CRISPR/Cas9 system in introducing a large provirus deletion in chicken PGCs. Our presented workflow is a cost-effective and rapid solution for screening the editing success in transfected PGCs. |
| first_indexed | 2024-04-11T10:01:48Z |
| format | Article |
| id | doaj.art-731dca4c8633414c96d99218e8b58ce9 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-11T10:01:48Z |
| publishDate | 2022-09-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-731dca4c8633414c96d99218e8b58ce92022-12-22T04:30:22ZengNature PortfolioScientific Reports2045-23222022-09-0112111310.1038/s41598-022-19861-7Quantitative analysis of CRISPR/Cas9-mediated provirus deletion in blue egg layer chicken PGCs by digital PCRStefanie Altgilbers0Claudia Dierks1Sabine Klein2Steffen Weigend3Wilfried A. Kues4Department of Biotechnology, Stem Cell Physiology, Institute of Farm Animal Genetics, Friedrich-Loeffler-InstitutDepartment of Breeding and Genetic Resources, Institute of Farm Animal Genetics, Friedrich-Loeffler-InstitutDepartment of Biotechnology, Stem Cell Physiology, Institute of Farm Animal Genetics, Friedrich-Loeffler-InstitutDepartment of Breeding and Genetic Resources, Institute of Farm Animal Genetics, Friedrich-Loeffler-InstitutDepartment of Biotechnology, Stem Cell Physiology, Institute of Farm Animal Genetics, Friedrich-Loeffler-InstitutAbstract Primordial germ cells (PGCs), the precursors of sperm and oocytes, pass on the genetic material to the next generation. The previously established culture system of chicken PGCs holds many possibilities for functional genomics studies and the rapid introduction of desired traits. Here, we established a CRISPR/Cas9-mediated genome editing protocol for the genetic modification of PGCs derived from chickens with blue eggshell color. The sequence targeted in the present report is a provirus (EAV-HP) insertion in the 5’-flanking region of the SLCO1B3 gene on chromosome 1 in Araucana chickens, which is supposedly responsible for the blue eggshell color. We designed pairs of guide RNAs (gRNAs) targeting the entire 4.2 kb provirus region. Following transfection of PGCs with the gRNA, genomic DNA was isolated and analyzed by mismatch cleavage assay (T7EI). For absolute quantification of the targeting efficiencies in homozygous blue-allele bearing PGCs a digital PCR was established, which revealed deletion efficiencies of 29% when the wildtype Cas9 was used, and 69% when a high-fidelity Cas9 variant was employed. Subsequent single cell dilutions of edited PGCs yielded 14 cell clones with homozygous deletion of the provirus. A digital PCR assay proved the complete absence of this provirus in cell clones. Thus, we demonstrated the high efficiency of the CRISPR/Cas9 system in introducing a large provirus deletion in chicken PGCs. Our presented workflow is a cost-effective and rapid solution for screening the editing success in transfected PGCs.https://doi.org/10.1038/s41598-022-19861-7 |
| spellingShingle | Stefanie Altgilbers Claudia Dierks Sabine Klein Steffen Weigend Wilfried A. Kues Quantitative analysis of CRISPR/Cas9-mediated provirus deletion in blue egg layer chicken PGCs by digital PCR Scientific Reports |
| title | Quantitative analysis of CRISPR/Cas9-mediated provirus deletion in blue egg layer chicken PGCs by digital PCR |
| title_full | Quantitative analysis of CRISPR/Cas9-mediated provirus deletion in blue egg layer chicken PGCs by digital PCR |
| title_fullStr | Quantitative analysis of CRISPR/Cas9-mediated provirus deletion in blue egg layer chicken PGCs by digital PCR |
| title_full_unstemmed | Quantitative analysis of CRISPR/Cas9-mediated provirus deletion in blue egg layer chicken PGCs by digital PCR |
| title_short | Quantitative analysis of CRISPR/Cas9-mediated provirus deletion in blue egg layer chicken PGCs by digital PCR |
| title_sort | quantitative analysis of crispr cas9 mediated provirus deletion in blue egg layer chicken pgcs by digital pcr |
| url | https://doi.org/10.1038/s41598-022-19861-7 |
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