Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis
CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use o...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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The Royal Society
2022-04-01
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| Series: | Open Biology |
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| Online Access: | https://royalsocietypublishing.org/doi/10.1098/rsob.210361 |
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| author | Vendula Horáčková Luboš Voleman Kari D. Hagen Markéta Petrů Martina Vinopalová Filip Weisz Natalia Janowicz Lenka Marková Alžběta Motyčková Vladimíra Najdrová Pavla Tůmová Scott C. Dawson Pavel Doležal |
| author_facet | Vendula Horáčková Luboš Voleman Kari D. Hagen Markéta Petrů Martina Vinopalová Filip Weisz Natalia Janowicz Lenka Marková Alžběta Motyčková Vladimíra Najdrová Pavla Tůmová Scott C. Dawson Pavel Doležal |
| author_sort | Vendula Horáčková |
| collection | DOAJ |
| description | CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis. |
| first_indexed | 2024-04-09T15:28:26Z |
| format | Article |
| id | doaj.art-d96837c111014c348aaa6b61ad7d506b |
| institution | Directory Open Access Journal |
| issn | 2046-2441 |
| language | English |
| last_indexed | 2024-04-09T15:28:26Z |
| publishDate | 2022-04-01 |
| publisher | The Royal Society |
| record_format | Article |
| series | Open Biology |
| spelling | doaj.art-d96837c111014c348aaa6b61ad7d506b2023-04-28T11:08:27ZengThe Royal SocietyOpen Biology2046-24412022-04-0112410.1098/rsob.210361Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalisVendula Horáčková0Luboš Voleman1Kari D. Hagen2Markéta Petrů3Martina Vinopalová4Filip Weisz5Natalia Janowicz6Lenka Marková7Alžběta Motyčková8Vladimíra Najdrová9Pavla Tůmová10Scott C. Dawson11Pavel Doležal12Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Praha, Czech RepublicDepartment of Parasitology, Faculty of Science, Charles University, BIOCEV, Praha, Czech RepublicDepartment of Microbiology and Molecular Genetics, College of Biological Sciences, UC Davis, Davis, CA, USADepartment of Parasitology, Faculty of Science, Charles University, BIOCEV, Praha, Czech RepublicDepartment of Parasitology, Faculty of Science, Charles University, BIOCEV, Praha, Czech RepublicInstitute of Immunology and Microbiology, First Faculty of Medicine, Charles University, Prague, Czech RepublicDepartment of Parasitology, Faculty of Science, Charles University, BIOCEV, Praha, Czech RepublicDepartment of Parasitology, Faculty of Science, Charles University, BIOCEV, Praha, Czech RepublicDepartment of Parasitology, Faculty of Science, Charles University, BIOCEV, Praha, Czech RepublicDepartment of Parasitology, Faculty of Science, Charles University, BIOCEV, Praha, Czech RepublicInstitute of Immunology and Microbiology, First Faculty of Medicine, Charles University, Prague, Czech RepublicDepartment of Microbiology and Molecular Genetics, College of Biological Sciences, UC Davis, Davis, CA, USADepartment of Parasitology, Faculty of Science, Charles University, BIOCEV, Praha, Czech RepublicCRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.https://royalsocietypublishing.org/doi/10.1098/rsob.210361GiardiaCRISPR/Cas9gene knockoutmultiploid |
| spellingShingle | Vendula Horáčková Luboš Voleman Kari D. Hagen Markéta Petrů Martina Vinopalová Filip Weisz Natalia Janowicz Lenka Marková Alžběta Motyčková Vladimíra Najdrová Pavla Tůmová Scott C. Dawson Pavel Doležal Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis Open Biology Giardia CRISPR/Cas9 gene knockout multiploid |
| title | Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis |
| title_full | Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis |
| title_fullStr | Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis |
| title_full_unstemmed | Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis |
| title_short | Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis |
| title_sort | efficient crispr cas9 mediated gene disruption in the tetraploid protist giardia intestinalis |
| topic | Giardia CRISPR/Cas9 gene knockout multiploid |
| url | https://royalsocietypublishing.org/doi/10.1098/rsob.210361 |
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