A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9
Abstract Background Adenine base editors (ABEs) are promising therapeutic gene editing tools that can efficiently convert targeted A•T to G•C base pairs in the genome. However, the large size of commonly used ABEs based on SpCas9 hinders its delivery in vivo using certain vectors such as adeno-assoc...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-07-01
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| Series: | BMC Biology |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12915-023-01644-9 |
| _version_ | 1797778737676156928 |
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| author | Yuqiang Qian Di Wang Wenchao Niu Ding Zhao Jinze Li Zhiquan Liu Xun Gao Yang Han Liangxue Lai Zhanjun Li |
| author_facet | Yuqiang Qian Di Wang Wenchao Niu Ding Zhao Jinze Li Zhiquan Liu Xun Gao Yang Han Liangxue Lai Zhanjun Li |
| author_sort | Yuqiang Qian |
| collection | DOAJ |
| description | Abstract Background Adenine base editors (ABEs) are promising therapeutic gene editing tools that can efficiently convert targeted A•T to G•C base pairs in the genome. However, the large size of commonly used ABEs based on SpCas9 hinders its delivery in vivo using certain vectors such as adeno-associated virus (AAV) during preclinical applications. Despite a number of approaches having previously been attempted to overcome that challenge, including split Cas9-derived and numerous domain-deleted versions of editors, whether base editor (BE) and prime editor (PE) systems can also allow deletion of those domains remains to be proven. In this study, we present a new small ABE (sABE) with significantly reduced size. Results We discovered that ABE8e can tolerate large single deletions in the REC2 (Δ174-296) and HNH (Δ786-855) domains of SpCas9, and these deletions can be stacked together to create a new sABE. The sABE showed higher precision than the original ABE8e, with proximally shifted protospacer adjacent motif (PAM) editing windows (A3- A15), and comparable editing efficiencies to 8e-SaCas9-KKH. The sABE system efficiently generated A-G mutations at disease-relevant loci (T1214C in GAA and A494G in MFN2) in HEK293T cells and several canonical Pcsk9 splice sites in N2a cells. Moreover, the sABE enabled in vivo delivery in a single adeno-associated virus (AAV) vector with slight efficiency. Furthermore, we also successfully edited the genome of mouse embryos by microinjecting mRNA and sgRNA of sABE system into zygotes. Conclusions We have developed a substantially smaller sABE system that expands the targeting scope and offers higher precision of genome editing. Our findings suggest that the sABE system holds great therapeutic potential in preclinical applications. |
| first_indexed | 2024-03-12T23:20:48Z |
| format | Article |
| id | doaj.art-f12c8b055be84e0a9a2bbc431c61a2ad |
| institution | Directory Open Access Journal |
| issn | 1741-7007 |
| language | English |
| last_indexed | 2024-03-12T23:20:48Z |
| publishDate | 2023-07-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biology |
| spelling | doaj.art-f12c8b055be84e0a9a2bbc431c61a2ad2023-07-16T11:27:57ZengBMCBMC Biology1741-70072023-07-0121111110.1186/s12915-023-01644-9A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9Yuqiang Qian0Di Wang1Wenchao Niu2Ding Zhao3Jinze Li4Zhiquan Liu5Xun Gao6Yang Han7Liangxue Lai8Zhanjun Li9Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin UniversityKey Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin UniversityKey Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin UniversityKey Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin UniversityKey Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin UniversityKey Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin UniversityKey Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin UniversityKey Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin UniversityKey Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin UniversityKey Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin UniversityAbstract Background Adenine base editors (ABEs) are promising therapeutic gene editing tools that can efficiently convert targeted A•T to G•C base pairs in the genome. However, the large size of commonly used ABEs based on SpCas9 hinders its delivery in vivo using certain vectors such as adeno-associated virus (AAV) during preclinical applications. Despite a number of approaches having previously been attempted to overcome that challenge, including split Cas9-derived and numerous domain-deleted versions of editors, whether base editor (BE) and prime editor (PE) systems can also allow deletion of those domains remains to be proven. In this study, we present a new small ABE (sABE) with significantly reduced size. Results We discovered that ABE8e can tolerate large single deletions in the REC2 (Δ174-296) and HNH (Δ786-855) domains of SpCas9, and these deletions can be stacked together to create a new sABE. The sABE showed higher precision than the original ABE8e, with proximally shifted protospacer adjacent motif (PAM) editing windows (A3- A15), and comparable editing efficiencies to 8e-SaCas9-KKH. The sABE system efficiently generated A-G mutations at disease-relevant loci (T1214C in GAA and A494G in MFN2) in HEK293T cells and several canonical Pcsk9 splice sites in N2a cells. Moreover, the sABE enabled in vivo delivery in a single adeno-associated virus (AAV) vector with slight efficiency. Furthermore, we also successfully edited the genome of mouse embryos by microinjecting mRNA and sgRNA of sABE system into zygotes. Conclusions We have developed a substantially smaller sABE system that expands the targeting scope and offers higher precision of genome editing. Our findings suggest that the sABE system holds great therapeutic potential in preclinical applications.https://doi.org/10.1186/s12915-023-01644-9CRISPR/Cas9Base editorABE8eReduce sizeAAV |
| spellingShingle | Yuqiang Qian Di Wang Wenchao Niu Ding Zhao Jinze Li Zhiquan Liu Xun Gao Yang Han Liangxue Lai Zhanjun Li A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9 BMC Biology CRISPR/Cas9 Base editor ABE8e Reduce size AAV |
| title | A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9 |
| title_full | A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9 |
| title_fullStr | A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9 |
| title_full_unstemmed | A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9 |
| title_short | A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9 |
| title_sort | new compact adenine base editor generated through deletion of hnh and rec2 domain of spcas9 |
| topic | CRISPR/Cas9 Base editor ABE8e Reduce size AAV |
| url | https://doi.org/10.1186/s12915-023-01644-9 |
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