Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope
Abstract Background Nme2ABE8e has been constructed and characterized as a compact, accurate adenine base editor with a less restrictive dinucleotide protospacer-adjacent motif (PAM: N4CC) but low editing efficiency at challenging loci in human cells. Here, we engineered a subset of domain-inlaid Nme...
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| Format: | Article |
| Language: | English |
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BMC
2023-11-01
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| Series: | BMC Biology |
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| Online Access: | https://doi.org/10.1186/s12915-023-01754-4 |
| _version_ | 1797629963790188544 |
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| author | Ding Zhao Xun Gao Jiale Zhou Jinze Li Yuqiang Qian Di Wang Wenchao Niu Tao Zhang Mingyang Hu Haoyang Xiong Liangxue Lai Zhanjun Li |
| author_facet | Ding Zhao Xun Gao Jiale Zhou Jinze Li Yuqiang Qian Di Wang Wenchao Niu Tao Zhang Mingyang Hu Haoyang Xiong Liangxue Lai Zhanjun Li |
| author_sort | Ding Zhao |
| collection | DOAJ |
| description | Abstract Background Nme2ABE8e has been constructed and characterized as a compact, accurate adenine base editor with a less restrictive dinucleotide protospacer-adjacent motif (PAM: N4CC) but low editing efficiency at challenging loci in human cells. Here, we engineered a subset of domain-inlaid Nme2Cas9 base editors to bring the deaminase domain closer to the nontarget strand to improve editing efficiency. Results Our results demonstrated that Nme2ABE8e-797 with adenine deaminase inserted between amino acids 797 and 798 has a significantly increased editing efficiency with a wide editing window ranging from 4 to 18 bases in mammalian cells, especially at the sites that were difficult to edit by Nme2ABE8e. In addition, by swapping the PAM-interacting domain of Nme2ABE8e-797 with that of SmuCas9 or introducing point mutations of eNme2-C in Nme2ABE8e-797, we created Nme2ABE8e-797Smu and Nme2ABE8e-797−C, respectively, which exhibited robust activities at a wide range of sites with N4CN PAMs in human cells. Moreover, the modified domain-inlaid Nme2ABE8e can efficiently restore or install disease-related loci in Neuro-2a cells and mice. Conclusions These novel Nme2ABE8es with increased on-target DNA editing and expanded PAM compatibility will expand the base editing toolset for efficient gene modification and therapeutic applications. |
| first_indexed | 2024-03-11T11:01:14Z |
| format | Article |
| id | doaj.art-876440371eb84c01918aa29e14cf99d1 |
| institution | Directory Open Access Journal |
| issn | 1741-7007 |
| language | English |
| last_indexed | 2024-03-11T11:01:14Z |
| publishDate | 2023-11-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biology |
| spelling | doaj.art-876440371eb84c01918aa29e14cf99d12023-11-12T12:30:18ZengBMCBMC Biology1741-70072023-11-0121111010.1186/s12915-023-01754-4Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scopeDing Zhao0Xun Gao1Jiale Zhou2Jinze Li3Yuqiang Qian4Di Wang5Wenchao Niu6Tao Zhang7Mingyang Hu8Haoyang Xiong9Liangxue Lai10Zhanjun Li11Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Veterinary Medicine, Jilin UniversityJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Veterinary Medicine, Jilin UniversityJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Veterinary Medicine, Jilin UniversityJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Veterinary Medicine, Jilin UniversityJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Veterinary Medicine, Jilin UniversityJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Veterinary Medicine, Jilin UniversityJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Veterinary Medicine, Jilin UniversityJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Veterinary Medicine, Jilin UniversityJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Veterinary Medicine, Jilin UniversityJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Veterinary Medicine, Jilin UniversityJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Veterinary Medicine, Jilin UniversityJilin Provincial Key Laboratory of Animal Embryo Engineering, College of Veterinary Medicine, Jilin UniversityAbstract Background Nme2ABE8e has been constructed and characterized as a compact, accurate adenine base editor with a less restrictive dinucleotide protospacer-adjacent motif (PAM: N4CC) but low editing efficiency at challenging loci in human cells. Here, we engineered a subset of domain-inlaid Nme2Cas9 base editors to bring the deaminase domain closer to the nontarget strand to improve editing efficiency. Results Our results demonstrated that Nme2ABE8e-797 with adenine deaminase inserted between amino acids 797 and 798 has a significantly increased editing efficiency with a wide editing window ranging from 4 to 18 bases in mammalian cells, especially at the sites that were difficult to edit by Nme2ABE8e. In addition, by swapping the PAM-interacting domain of Nme2ABE8e-797 with that of SmuCas9 or introducing point mutations of eNme2-C in Nme2ABE8e-797, we created Nme2ABE8e-797Smu and Nme2ABE8e-797−C, respectively, which exhibited robust activities at a wide range of sites with N4CN PAMs in human cells. Moreover, the modified domain-inlaid Nme2ABE8e can efficiently restore or install disease-related loci in Neuro-2a cells and mice. Conclusions These novel Nme2ABE8es with increased on-target DNA editing and expanded PAM compatibility will expand the base editing toolset for efficient gene modification and therapeutic applications.https://doi.org/10.1186/s12915-023-01754-4Adenine base editorsInlaid domainNme2ABE8e-797Nme2ABE8e-797SmuNme2ABE8e-797−C |
| spellingShingle | Ding Zhao Xun Gao Jiale Zhou Jinze Li Yuqiang Qian Di Wang Wenchao Niu Tao Zhang Mingyang Hu Haoyang Xiong Liangxue Lai Zhanjun Li Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope BMC Biology Adenine base editors Inlaid domain Nme2ABE8e-797 Nme2ABE8e-797Smu Nme2ABE8e-797−C |
| title | Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope |
| title_full | Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope |
| title_fullStr | Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope |
| title_full_unstemmed | Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope |
| title_short | Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope |
| title_sort | engineered domain inlaid nme2cas9 adenine base editors with increased on target dna editing and targeting scope |
| topic | Adenine base editors Inlaid domain Nme2ABE8e-797 Nme2ABE8e-797Smu Nme2ABE8e-797−C |
| url | https://doi.org/10.1186/s12915-023-01754-4 |
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