Enhancing Prime Editing Efficiency Through Modulation of Methylation on the Newly Synthesized DNA Strand and Prolonged Expression
Abstract Prime editors (PEs) have emerged as transformative tools for precision genome engineering, yet their broader application remains constrained by incomplete understanding of repair mechanisms. In this study, it is found that an increase in the methylation level of the CpG sequence on the newl...
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Wiley
2025-05-01
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| Series: | Advanced Science |
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| Online Access: | https://doi.org/10.1002/advs.202417790 |
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| author | Xiaosong Han Xianghua Xu Youcai Xiong Guangxing Zhao Ruigao He Yinyu Su Sheng Li Changzhi Zhao Xiaoning Xi Yunxia Zhao Xuewen Xu Shengsong Xie Heng Wang Xinyun Li Shuhong Zhao Jinxue Ruan |
| author_facet | Xiaosong Han Xianghua Xu Youcai Xiong Guangxing Zhao Ruigao He Yinyu Su Sheng Li Changzhi Zhao Xiaoning Xi Yunxia Zhao Xuewen Xu Shengsong Xie Heng Wang Xinyun Li Shuhong Zhao Jinxue Ruan |
| author_sort | Xiaosong Han |
| collection | DOAJ |
| description | Abstract Prime editors (PEs) have emerged as transformative tools for precision genome engineering, yet their broader application remains constrained by incomplete understanding of repair mechanisms. In this study, it is found that an increase in the methylation level of the CpG sequence on the newly generated strand can increase PE efficiency and that de novo DNA methyltransferases (DNMT3A/3B) are involved in the PE repair pathway. On the basis of these novel findings, the development of an episomal element‐driven PE system (epiPE) achieved through the use of EBNA1/oriP are presented, which increases methylation levels around target sites and prolongs PE expression. A comparative analysis with canonical PE systems, including PE2, lentiPE2, and PE4max, reveals that the epiPE2 system significantly enhances editing efficiency while maintaining minimal insertion and deletion (indels) rates. Specifically, comparing to PE2, the epiPE2 system demonstrated an efficiency enhancement of 2.0 to 38.2‐fold. In addition, the epiPE2 system is capable of efficient multiplex precise gene editing at up to 10 genetic loci in human cells. In conclusion, this findings increase the understanding of the PE repair mechanism, and presents the epiPE2 system as an efficient and multiplex‐capable prime editing tool with potential applications in both basic research and translational studies. |
| format | Article |
| id | doaj-art-0d8bc6de59254502b938c7e20c3bcb33 |
| institution | Kabale University |
| issn | 2198-3844 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Wiley |
| record_format | Article |
| series | Advanced Science |
| spelling | doaj-art-0d8bc6de59254502b938c7e20c3bcb332025-08-20T03:48:47ZengWileyAdvanced Science2198-38442025-05-011217n/an/a10.1002/advs.202417790Enhancing Prime Editing Efficiency Through Modulation of Methylation on the Newly Synthesized DNA Strand and Prolonged ExpressionXiaosong Han0Xianghua Xu1Youcai Xiong2Guangxing Zhao3Ruigao He4Yinyu Su5Sheng Li6Changzhi Zhao7Xiaoning Xi8Yunxia Zhao9Xuewen Xu10Shengsong Xie11Heng Wang12Xinyun Li13Shuhong Zhao14Jinxue Ruan15Key Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaKey Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaKey Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaKey Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaKey Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaKey Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaKey Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaKey Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaKey Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaKey Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaKey Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaKey Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaKey Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaKey Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaKey Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaKey Laboratory of Agricultural Animal Genetics Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs Huazhong Agricultural University Wuhan 430070 P. R. ChinaAbstract Prime editors (PEs) have emerged as transformative tools for precision genome engineering, yet their broader application remains constrained by incomplete understanding of repair mechanisms. In this study, it is found that an increase in the methylation level of the CpG sequence on the newly generated strand can increase PE efficiency and that de novo DNA methyltransferases (DNMT3A/3B) are involved in the PE repair pathway. On the basis of these novel findings, the development of an episomal element‐driven PE system (epiPE) achieved through the use of EBNA1/oriP are presented, which increases methylation levels around target sites and prolongs PE expression. A comparative analysis with canonical PE systems, including PE2, lentiPE2, and PE4max, reveals that the epiPE2 system significantly enhances editing efficiency while maintaining minimal insertion and deletion (indels) rates. Specifically, comparing to PE2, the epiPE2 system demonstrated an efficiency enhancement of 2.0 to 38.2‐fold. In addition, the epiPE2 system is capable of efficient multiplex precise gene editing at up to 10 genetic loci in human cells. In conclusion, this findings increase the understanding of the PE repair mechanism, and presents the epiPE2 system as an efficient and multiplex‐capable prime editing tool with potential applications in both basic research and translational studies.https://doi.org/10.1002/advs.202417790EBNA1/oriPgene editingmethylationmultiplexprime editor |
| spellingShingle | Xiaosong Han Xianghua Xu Youcai Xiong Guangxing Zhao Ruigao He Yinyu Su Sheng Li Changzhi Zhao Xiaoning Xi Yunxia Zhao Xuewen Xu Shengsong Xie Heng Wang Xinyun Li Shuhong Zhao Jinxue Ruan Enhancing Prime Editing Efficiency Through Modulation of Methylation on the Newly Synthesized DNA Strand and Prolonged Expression Advanced Science EBNA1/oriP gene editing methylation multiplex prime editor |
| title | Enhancing Prime Editing Efficiency Through Modulation of Methylation on the Newly Synthesized DNA Strand and Prolonged Expression |
| title_full | Enhancing Prime Editing Efficiency Through Modulation of Methylation on the Newly Synthesized DNA Strand and Prolonged Expression |
| title_fullStr | Enhancing Prime Editing Efficiency Through Modulation of Methylation on the Newly Synthesized DNA Strand and Prolonged Expression |
| title_full_unstemmed | Enhancing Prime Editing Efficiency Through Modulation of Methylation on the Newly Synthesized DNA Strand and Prolonged Expression |
| title_short | Enhancing Prime Editing Efficiency Through Modulation of Methylation on the Newly Synthesized DNA Strand and Prolonged Expression |
| title_sort | enhancing prime editing efficiency through modulation of methylation on the newly synthesized dna strand and prolonged expression |
| topic | EBNA1/oriP gene editing methylation multiplex prime editor |
| url | https://doi.org/10.1002/advs.202417790 |
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