Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing
The naturally occurring prokaryotic CRISPR-Cas systems provide valuable resources for the development of new genome-editing tools. However, the majority of prokaryotic Cas nucleases exhibit poor editing efficiency in mammalian cells, which significantly limits their utility. Here, we have developed...
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| Format: | Article |
| Language: | English |
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Elsevier
2022-07-01
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| Series: | The Innovation |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666675822000601 |
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| author | Yangcan Chen Yanping Hu Xinge Wang Shengqiu Luo Ning Yang Yi Chen Zhikun Li Qi Zhou Wei Li |
| author_facet | Yangcan Chen Yanping Hu Xinge Wang Shengqiu Luo Ning Yang Yi Chen Zhikun Li Qi Zhou Wei Li |
| author_sort | Yangcan Chen |
| collection | DOAJ |
| description | The naturally occurring prokaryotic CRISPR-Cas systems provide valuable resources for the development of new genome-editing tools. However, the majority of prokaryotic Cas nucleases exhibit poor editing efficiency in mammalian cells, which significantly limits their utility. Here, we have developed a method termed Improving Editing Activity by Synergistic Engineering (MIDAS). This method exerts a synergistic effect to improve mammalian genome-editing efficiency of a wide range of CRISPR-Cas systems by enhancing the interactions between Cas nuclease with the protospacer adjacent motif (PAM) and the single-stranded DNA (ssDNA) substrate in the catalytic pocket simultaneously. MIDAS robustly and significantly increased the gene-editing efficiency of Cas12i, Cas12b, and CasX in human cells. Notably, a Cas12i variant, Cas12iMax, exhibited robust activity with a very broad PAM range (NTNN, NNTN, NAAN, and NCAN) and higher efficiency than the current widely used Cas nucleases. A high-fidelity version of Cas12iMax (Cas12iHiFi) has been further engineered to minimize off-target effects. Our work provides an expandable and efficacious method for engineering Cas nucleases for robust mammalian genome editing. |
| first_indexed | 2024-04-12T18:11:16Z |
| format | Article |
| id | doaj.art-9bfde642d50949a0aa4456a52f212fba |
| institution | Directory Open Access Journal |
| issn | 2666-6758 |
| language | English |
| last_indexed | 2024-04-12T18:11:16Z |
| publishDate | 2022-07-01 |
| publisher | Elsevier |
| record_format | Article |
| series | The Innovation |
| spelling | doaj.art-9bfde642d50949a0aa4456a52f212fba2022-12-22T03:21:50ZengElsevierThe Innovation2666-67582022-07-0134100264Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editingYangcan Chen0Yanping Hu1Xinge Wang2Shengqiu Luo3Ning Yang4Yi Chen5Zhikun Li6Qi Zhou7Wei Li8State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing 100101, ChinaState Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing 100101, ChinaState Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing 100101, ChinaState Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing 100101, ChinaState Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing 100101, ChinaState Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing 100101, ChinaState Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing 100101, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, ChinaState Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing 100101, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China; Corresponding authorState Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing 100101, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China; Corresponding authorThe naturally occurring prokaryotic CRISPR-Cas systems provide valuable resources for the development of new genome-editing tools. However, the majority of prokaryotic Cas nucleases exhibit poor editing efficiency in mammalian cells, which significantly limits their utility. Here, we have developed a method termed Improving Editing Activity by Synergistic Engineering (MIDAS). This method exerts a synergistic effect to improve mammalian genome-editing efficiency of a wide range of CRISPR-Cas systems by enhancing the interactions between Cas nuclease with the protospacer adjacent motif (PAM) and the single-stranded DNA (ssDNA) substrate in the catalytic pocket simultaneously. MIDAS robustly and significantly increased the gene-editing efficiency of Cas12i, Cas12b, and CasX in human cells. Notably, a Cas12i variant, Cas12iMax, exhibited robust activity with a very broad PAM range (NTNN, NNTN, NAAN, and NCAN) and higher efficiency than the current widely used Cas nucleases. A high-fidelity version of Cas12iMax (Cas12iHiFi) has been further engineered to minimize off-target effects. Our work provides an expandable and efficacious method for engineering Cas nucleases for robust mammalian genome editing.http://www.sciencedirect.com/science/article/pii/S2666675822000601protein engineeringgenome editingCRISPR-Cas12isimple PAM requirementhigh fidelity |
| spellingShingle | Yangcan Chen Yanping Hu Xinge Wang Shengqiu Luo Ning Yang Yi Chen Zhikun Li Qi Zhou Wei Li Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing The Innovation protein engineering genome editing CRISPR-Cas12i simple PAM requirement high fidelity |
| title | Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing |
| title_full | Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing |
| title_fullStr | Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing |
| title_full_unstemmed | Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing |
| title_short | Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing |
| title_sort | synergistic engineering of crispr cas nucleases enables robust mammalian genome editing |
| topic | protein engineering genome editing CRISPR-Cas12i simple PAM requirement high fidelity |
| url | http://www.sciencedirect.com/science/article/pii/S2666675822000601 |
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