A strategy for Cas13 miniaturization based on the structure and AlphaFold
Abstract The small size of the Cas nuclease fused with various effector domains enables a broad range of function. Although there are several ways of reducing the size of the Cas nuclease complex, no efficient or generalizable method has been demonstrated to achieve protein miniaturization. In this...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-09-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-023-41320-8 |
| _version_ | 1797558318304067584 |
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| author | Feiyu Zhao Tao Zhang Xiaodi Sun Xiyun Zhang Letong Chen Hejun Wang Jinze Li Peng Fan Liangxue Lai Tingting Sui Zhanjun Li |
| author_facet | Feiyu Zhao Tao Zhang Xiaodi Sun Xiyun Zhang Letong Chen Hejun Wang Jinze Li Peng Fan Liangxue Lai Tingting Sui Zhanjun Li |
| author_sort | Feiyu Zhao |
| collection | DOAJ |
| description | Abstract The small size of the Cas nuclease fused with various effector domains enables a broad range of function. Although there are several ways of reducing the size of the Cas nuclease complex, no efficient or generalizable method has been demonstrated to achieve protein miniaturization. In this study, we establish an Interaction, Dynamics and Conservation (IDC) strategy for protein miniaturization and generate five compact variants of Cas13 with full RNA binding and cleavage activity comparable the wild-type enzymes based on a combination of IDC strategy and AlphaFold2. In addition, we construct an RNA base editor, mini-Vx, and a single AAV (adeno-associated virus) carrying a mini-RfxCas13d and crRNA expression cassette, which individually shows efficient conversion rate and RNA-knockdown activity. In summary, these findings highlight a feasible strategy for generating downsized CRISPR/Cas13 systems based on structure predicted by AlphaFold2, enabling targeted degradation of RNAs and RNA editing for basic research and therapeutic applications. |
| first_indexed | 2024-03-10T17:29:47Z |
| format | Article |
| id | doaj.art-805df6dda3454999869b65dae19f19a3 |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-03-10T17:29:47Z |
| publishDate | 2023-09-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-805df6dda3454999869b65dae19f19a32023-11-20T10:04:21ZengNature PortfolioNature Communications2041-17232023-09-0114111310.1038/s41467-023-41320-8A strategy for Cas13 miniaturization based on the structure and AlphaFoldFeiyu Zhao0Tao Zhang1Xiaodi Sun2Xiyun Zhang3Letong Chen4Hejun Wang5Jinze Li6Peng Fan7Liangxue Lai8Tingting Sui9Zhanjun Li10State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin UniversityState Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin UniversityState Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin UniversityState Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin UniversityState Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin UniversityState Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin UniversityState Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin UniversityState Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin UniversityState Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin UniversityState Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin UniversityState Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin UniversityAbstract The small size of the Cas nuclease fused with various effector domains enables a broad range of function. Although there are several ways of reducing the size of the Cas nuclease complex, no efficient or generalizable method has been demonstrated to achieve protein miniaturization. In this study, we establish an Interaction, Dynamics and Conservation (IDC) strategy for protein miniaturization and generate five compact variants of Cas13 with full RNA binding and cleavage activity comparable the wild-type enzymes based on a combination of IDC strategy and AlphaFold2. In addition, we construct an RNA base editor, mini-Vx, and a single AAV (adeno-associated virus) carrying a mini-RfxCas13d and crRNA expression cassette, which individually shows efficient conversion rate and RNA-knockdown activity. In summary, these findings highlight a feasible strategy for generating downsized CRISPR/Cas13 systems based on structure predicted by AlphaFold2, enabling targeted degradation of RNAs and RNA editing for basic research and therapeutic applications.https://doi.org/10.1038/s41467-023-41320-8 |
| spellingShingle | Feiyu Zhao Tao Zhang Xiaodi Sun Xiyun Zhang Letong Chen Hejun Wang Jinze Li Peng Fan Liangxue Lai Tingting Sui Zhanjun Li A strategy for Cas13 miniaturization based on the structure and AlphaFold Nature Communications |
| title | A strategy for Cas13 miniaturization based on the structure and AlphaFold |
| title_full | A strategy for Cas13 miniaturization based on the structure and AlphaFold |
| title_fullStr | A strategy for Cas13 miniaturization based on the structure and AlphaFold |
| title_full_unstemmed | A strategy for Cas13 miniaturization based on the structure and AlphaFold |
| title_short | A strategy for Cas13 miniaturization based on the structure and AlphaFold |
| title_sort | strategy for cas13 miniaturization based on the structure and alphafold |
| url | https://doi.org/10.1038/s41467-023-41320-8 |
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