Engineering of SauriCas9 with enhanced specificity
SauriCas9 is a compact Cas9 nuclease showing promise for in vivo therapeutic applications. However, concerns about off-target effects necessitated improvements in specificity. We addressed this by introducing mutations to eliminate polar contacts between Cas9 and the target DNA, resulting in the Sau...
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| Format: | Article |
| Language: | English |
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Elsevier
2025-03-01
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| Series: | Molecular Therapy: Nucleic Acids |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253125000095 |
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| author | Xiaoqi Zhang Chen Tao Miaomiao Li Sufang Zhang Puping Liang Yan Huang Huihui Liu Yongming Wang |
| author_facet | Xiaoqi Zhang Chen Tao Miaomiao Li Sufang Zhang Puping Liang Yan Huang Huihui Liu Yongming Wang |
| author_sort | Xiaoqi Zhang |
| collection | DOAJ |
| description | SauriCas9 is a compact Cas9 nuclease showing promise for in vivo therapeutic applications. However, concerns about off-target effects necessitated improvements in specificity. We addressed this by introducing mutations to eliminate polar contacts between Cas9 and the target DNA, resulting in the SauriCas9-R253A variant with enhanced specificity. To validate its efficacy, we employed SauriCas9-R253A to disrupt three genes (B2M, TRAC, and PDCD1), a strategy integral to the development of allogeneic chimeric antigen receptor T cell (CAR-T) therapies. Our results demonstrated that the most efficient single-guide RNAs for SauriCas9-R253A exhibited comparable activity to SpCas9 and showed no detectable off-target effects in the disruption of these genes, highlighting its therapeutic potential. |
| format | Article |
| id | doaj-art-69c8ab0cb20f4e278450a056e9adae01 |
| institution | Kabale University |
| issn | 2162-2531 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj-art-69c8ab0cb20f4e278450a056e9adae012025-02-12T05:30:59ZengElsevierMolecular Therapy: Nucleic Acids2162-25312025-03-01361102455Engineering of SauriCas9 with enhanced specificityXiaoqi Zhang0Chen Tao1Miaomiao Li2Sufang Zhang3Puping Liang4Yan Huang5Huihui Liu6Yongming Wang7Center for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, School of Life Sciences, Fudan University, Shanghai 200438, ChinaCenter for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, School of Life Sciences, Fudan University, Shanghai 200438, ChinaCenter for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, School of Life Sciences, Fudan University, Shanghai 200438, ChinaKey Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing 100091, ChinaState Key Laboratory of Biocontrol, MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, ChinaState Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University, Shanghai 200433, China; Corresponding author: Yan Huang, State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University, Shanghai 200433, China.National Permanent Scientific Research Base for Warm Temperate Zone Forestry of Jiulong Mountain in Beijing, Experimental Center of Forestry in North China, CAF, Beijing 102300, China; Corresponding author: Huihui Liu, National Permanent Scientific Research Base for Warm Temperate Zone Forestry of Jiulong Mountain in Beijing, Experimental Center of Forestry in North China, CAF, Beijing 102300, China.Center for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, School of Life Sciences, Fudan University, Shanghai 200438, China; State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University, Shanghai 200433, China; Corresponding author: Yongming Wang, Center for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, School of Life Sciences, Fudan University, Shanghai 200438, China.SauriCas9 is a compact Cas9 nuclease showing promise for in vivo therapeutic applications. However, concerns about off-target effects necessitated improvements in specificity. We addressed this by introducing mutations to eliminate polar contacts between Cas9 and the target DNA, resulting in the SauriCas9-R253A variant with enhanced specificity. To validate its efficacy, we employed SauriCas9-R253A to disrupt three genes (B2M, TRAC, and PDCD1), a strategy integral to the development of allogeneic chimeric antigen receptor T cell (CAR-T) therapies. Our results demonstrated that the most efficient single-guide RNAs for SauriCas9-R253A exhibited comparable activity to SpCas9 and showed no detectable off-target effects in the disruption of these genes, highlighting its therapeutic potential.http://www.sciencedirect.com/science/article/pii/S2162253125000095MT: RNA/DNA Editinggene editingCRISPR-Cascompact Cas9SauriCas9-R253Aspecificity |
| spellingShingle | Xiaoqi Zhang Chen Tao Miaomiao Li Sufang Zhang Puping Liang Yan Huang Huihui Liu Yongming Wang Engineering of SauriCas9 with enhanced specificity Molecular Therapy: Nucleic Acids MT: RNA/DNA Editing gene editing CRISPR-Cas compact Cas9 SauriCas9-R253A specificity |
| title | Engineering of SauriCas9 with enhanced specificity |
| title_full | Engineering of SauriCas9 with enhanced specificity |
| title_fullStr | Engineering of SauriCas9 with enhanced specificity |
| title_full_unstemmed | Engineering of SauriCas9 with enhanced specificity |
| title_short | Engineering of SauriCas9 with enhanced specificity |
| title_sort | engineering of sauricas9 with enhanced specificity |
| topic | MT: RNA/DNA Editing gene editing CRISPR-Cas compact Cas9 SauriCas9-R253A specificity |
| url | http://www.sciencedirect.com/science/article/pii/S2162253125000095 |
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