Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from <i>Ruminococcus bromii</i>
Type V Cas12a nucleases are DNA editors working in a wide temperature range and using expanded protospacer-adjacent motifs (PAMs). Though they are widely used, there is still a demand for discovering new ones. Here, we demonstrate a novel ortholog from <i>Ruminococcus bromii</i> sp. enti...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-08-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/16/9289 |
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| author | Ruslan Vasilev Natalia Gunitseva Regina Shebanova Aleksei Korzhenkov Anna Vlaskina Marta Evteeva Irina Polushkina Natalia Nikitchina Stepan Toshchakov Piotr Kamenski Maxim Patrushev Ilya Mazunin |
| author_facet | Ruslan Vasilev Natalia Gunitseva Regina Shebanova Aleksei Korzhenkov Anna Vlaskina Marta Evteeva Irina Polushkina Natalia Nikitchina Stepan Toshchakov Piotr Kamenski Maxim Patrushev Ilya Mazunin |
| author_sort | Ruslan Vasilev |
| collection | DOAJ |
| description | Type V Cas12a nucleases are DNA editors working in a wide temperature range and using expanded protospacer-adjacent motifs (PAMs). Though they are widely used, there is still a demand for discovering new ones. Here, we demonstrate a novel ortholog from <i>Ruminococcus bromii</i> sp. entitled RbCas12a, which is able to efficiently cleave target DNA templates, using the particularly high accessibility of PAM 5′-YYN and a relatively wide temperature range from 20 °C to 42 °C. In comparison to <i>Acidaminococcus</i> sp. (AsCas12a) nuclease, RbCas12a is capable of processing DNA more efficiently, and can be active upon being charged by spacer-only RNA at lower concentrations in vitro. We show that the human-optimized RbCas12a nuclease is also active in mammalian cells, and can be applied for efficient deletion incorporation into the human genome. Given the advantageous properties of RbCas12a, this enzyme shows potential for clinical and biotechnological applications within the field of genome editing. |
| first_indexed | 2024-03-09T04:18:42Z |
| format | Article |
| id | doaj.art-24446ab8ee9e4b39a0d4310bb3bc64dd |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T04:18:42Z |
| publishDate | 2022-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-24446ab8ee9e4b39a0d4310bb3bc64dd2023-12-03T13:49:38ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-08-012316928910.3390/ijms23169289Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from <i>Ruminococcus bromii</i>Ruslan Vasilev0Natalia Gunitseva1Regina Shebanova2Aleksei Korzhenkov3Anna Vlaskina4Marta Evteeva5Irina Polushkina6Natalia Nikitchina7Stepan Toshchakov8Piotr Kamenski9Maxim Patrushev10Ilya Mazunin11Kurchatov Genomics Center, National Research Center “Kurchatov Institute”, 123098 Moscow, RussiaKurchatov Genomics Center, National Research Center “Kurchatov Institute”, 123098 Moscow, RussiaCenter of Life Sciences, Skolkovo Institute of Science and Technology, 143026 Moscow, RussiaKurchatov Genomics Center, National Research Center “Kurchatov Institute”, 123098 Moscow, RussiaKurchatov Genomics Center, National Research Center “Kurchatov Institute”, 123098 Moscow, RussiaKurchatov Genomics Center, National Research Center “Kurchatov Institute”, 123098 Moscow, RussiaKurchatov Genomics Center, National Research Center “Kurchatov Institute”, 123098 Moscow, RussiaCenter of Life Sciences, Skolkovo Institute of Science and Technology, 143026 Moscow, RussiaKurchatov Genomics Center, National Research Center “Kurchatov Institute”, 123098 Moscow, RussiaFaculty of Biology, Lomonosov Moscow State University, 119991 Moscow, RussiaKurchatov Genomics Center, National Research Center “Kurchatov Institute”, 123098 Moscow, RussiaCenter of Life Sciences, Skolkovo Institute of Science and Technology, 143026 Moscow, RussiaType V Cas12a nucleases are DNA editors working in a wide temperature range and using expanded protospacer-adjacent motifs (PAMs). Though they are widely used, there is still a demand for discovering new ones. Here, we demonstrate a novel ortholog from <i>Ruminococcus bromii</i> sp. entitled RbCas12a, which is able to efficiently cleave target DNA templates, using the particularly high accessibility of PAM 5′-YYN and a relatively wide temperature range from 20 °C to 42 °C. In comparison to <i>Acidaminococcus</i> sp. (AsCas12a) nuclease, RbCas12a is capable of processing DNA more efficiently, and can be active upon being charged by spacer-only RNA at lower concentrations in vitro. We show that the human-optimized RbCas12a nuclease is also active in mammalian cells, and can be applied for efficient deletion incorporation into the human genome. Given the advantageous properties of RbCas12a, this enzyme shows potential for clinical and biotechnological applications within the field of genome editing.https://www.mdpi.com/1422-0067/23/16/9289Cas endonucleaseCRISPRgenome editingmammalian cellssite-directed mutagenesis |
| spellingShingle | Ruslan Vasilev Natalia Gunitseva Regina Shebanova Aleksei Korzhenkov Anna Vlaskina Marta Evteeva Irina Polushkina Natalia Nikitchina Stepan Toshchakov Piotr Kamenski Maxim Patrushev Ilya Mazunin Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from <i>Ruminococcus bromii</i> International Journal of Molecular Sciences Cas endonuclease CRISPR genome editing mammalian cells site-directed mutagenesis |
| title | Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from <i>Ruminococcus bromii</i> |
| title_full | Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from <i>Ruminococcus bromii</i> |
| title_fullStr | Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from <i>Ruminococcus bromii</i> |
| title_full_unstemmed | Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from <i>Ruminococcus bromii</i> |
| title_short | Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from <i>Ruminococcus bromii</i> |
| title_sort | targeted modification of mammalian dna by a novel type v cas12a endonuclease from i ruminococcus bromii i |
| topic | Cas endonuclease CRISPR genome editing mammalian cells site-directed mutagenesis |
| url | https://www.mdpi.com/1422-0067/23/16/9289 |
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