Mismatch prime editing gRNA increased efficiency and reduced indels
Abstract Prime editing enables precise and efficient genome editing, but its efficacy is hindered by pegRNA’s 3’ extension, forming secondary structures due to high complementarity with the protospacer. The continuous presence of the prime editing system also leads to unintended indel formation, rai...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-01-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-024-55578-z |
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| _version_ | 1841559317004681216 |
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| author | Jidong Fei Dongdong Zhao Caiyi Pang Ju Li Siwei Li Wentao Qiao Juan Tan Changhao Bi Xueli Zhang |
| author_facet | Jidong Fei Dongdong Zhao Caiyi Pang Ju Li Siwei Li Wentao Qiao Juan Tan Changhao Bi Xueli Zhang |
| author_sort | Jidong Fei |
| collection | DOAJ |
| description | Abstract Prime editing enables precise and efficient genome editing, but its efficacy is hindered by pegRNA’s 3’ extension, forming secondary structures due to high complementarity with the protospacer. The continuous presence of the prime editing system also leads to unintended indel formation, raising safety concerns for therapeutic applications. To address these challenges, we develop a mismatched pegRNA (mpegRNA) strategy that introduces mismatched bases into the pegRNA protospacer, reducing complementarity and secondary structure formation, and preventing sustained activity. Our findings show that mpegRNA enhances editing efficiency by up to 2.3 times and reduces indel levels by 76.5% without compromising performance. Combining mpegRNA with epegRNA further increases efficiency up to 14-fold, or 2.4-fold in PE4max/PE5max systems, underscoring its potential in research and therapy. AlphaFold 3 analysis suggests that the optimal mpegRNA structure contributes significantly to improved editing outcomes. Overall, mpegRNA advances prime editing technology, improving efficiency while reducing indels. |
| format | Article |
| id | doaj-art-cdfbc83638364ff8a96453e313fbf93b |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-cdfbc83638364ff8a96453e313fbf93b2025-01-05T12:37:44ZengNature PortfolioNature Communications2041-17232025-01-0116111010.1038/s41467-024-55578-zMismatch prime editing gRNA increased efficiency and reduced indelsJidong Fei0Dongdong Zhao1Caiyi Pang2Ju Li3Siwei Li4Wentao Qiao5Juan Tan6Changhao Bi7Xueli Zhang8College of Life Science, Tianjin Normal UniversityTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesCollege of Life Science, Tianjin Normal UniversityTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesKey Laboratory of Molecular Microbiology and Technology, Ministry of Education, College of Life Sciences, Nankai UniversityKey Laboratory of Molecular Microbiology and Technology, Ministry of Education, College of Life Sciences, Nankai UniversityTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesAbstract Prime editing enables precise and efficient genome editing, but its efficacy is hindered by pegRNA’s 3’ extension, forming secondary structures due to high complementarity with the protospacer. The continuous presence of the prime editing system also leads to unintended indel formation, raising safety concerns for therapeutic applications. To address these challenges, we develop a mismatched pegRNA (mpegRNA) strategy that introduces mismatched bases into the pegRNA protospacer, reducing complementarity and secondary structure formation, and preventing sustained activity. Our findings show that mpegRNA enhances editing efficiency by up to 2.3 times and reduces indel levels by 76.5% without compromising performance. Combining mpegRNA with epegRNA further increases efficiency up to 14-fold, or 2.4-fold in PE4max/PE5max systems, underscoring its potential in research and therapy. AlphaFold 3 analysis suggests that the optimal mpegRNA structure contributes significantly to improved editing outcomes. Overall, mpegRNA advances prime editing technology, improving efficiency while reducing indels.https://doi.org/10.1038/s41467-024-55578-z |
| spellingShingle | Jidong Fei Dongdong Zhao Caiyi Pang Ju Li Siwei Li Wentao Qiao Juan Tan Changhao Bi Xueli Zhang Mismatch prime editing gRNA increased efficiency and reduced indels Nature Communications |
| title | Mismatch prime editing gRNA increased efficiency and reduced indels |
| title_full | Mismatch prime editing gRNA increased efficiency and reduced indels |
| title_fullStr | Mismatch prime editing gRNA increased efficiency and reduced indels |
| title_full_unstemmed | Mismatch prime editing gRNA increased efficiency and reduced indels |
| title_short | Mismatch prime editing gRNA increased efficiency and reduced indels |
| title_sort | mismatch prime editing grna increased efficiency and reduced indels |
| url | https://doi.org/10.1038/s41467-024-55578-z |
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