Modified pegRNAs mitigate scaffold-derived prime editing by-products
Abstract Prime editors (PEs) employ reverse transcriptase (RT) to install genomic edits using a template within the prime editing guide RNA (pegRNA). RT creates a 3’ genomic flap containing the intended edit. However, reverse transcription can continue beyond the template, incorporating the pegRNA s...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-04-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-58653-1 |
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| author | Panagiotis Antoniou Louis Dacquay Hanna Mårtensson Katja Madeyski-Bengtson Anna-Lena Loyd Anna Shiriaeva Euan Gordon Salman Mustfa George Thom Pei-Pei Hsieh Saša Šviković Mike Firth Nina Akrap Marcello Maresca Martin Peterka |
| author_facet | Panagiotis Antoniou Louis Dacquay Hanna Mårtensson Katja Madeyski-Bengtson Anna-Lena Loyd Anna Shiriaeva Euan Gordon Salman Mustfa George Thom Pei-Pei Hsieh Saša Šviković Mike Firth Nina Akrap Marcello Maresca Martin Peterka |
| author_sort | Panagiotis Antoniou |
| collection | DOAJ |
| description | Abstract Prime editors (PEs) employ reverse transcriptase (RT) to install genomic edits using a template within the prime editing guide RNA (pegRNA). RT creates a 3’ genomic flap containing the intended edit. However, reverse transcription can continue beyond the template, incorporating the pegRNA scaffold sequence into the 3’ flap. These scaffold-derived by-products can be installed alongside the intended edit, reducing prime editing precision. Here, we develop a method that prevents RT from accessing the scaffold, thereby mitigating such by-products. We demonstrate that an internal abasic spacer or 2’-O-methylation within the pegRNAs terminates RT at the end of the template. This prevents scaffold-derived sequences from being incorporated into the target locus. We benchmark these pegRNAs in different cell types and demonstrate that they can be used with processive PEs such as PE6d or PE**. Our findings provide a simple approach to mitigate a common prime editing by-product and improve prime editing precision. |
| format | Article |
| id | doaj-art-2c66a9ebc0564c0fb3808c4f6e52a16b |
| institution | OA Journals |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-2c66a9ebc0564c0fb3808c4f6e52a16b2025-08-20T02:11:47ZengNature PortfolioNature Communications2041-17232025-04-0116111110.1038/s41467-025-58653-1Modified pegRNAs mitigate scaffold-derived prime editing by-productsPanagiotis Antoniou0Louis Dacquay1Hanna Mårtensson2Katja Madeyski-Bengtson3Anna-Lena Loyd4Anna Shiriaeva5Euan Gordon6Salman Mustfa7George Thom8Pei-Pei Hsieh9Saša Šviković10Mike Firth11Nina Akrap12Marcello Maresca13Martin Peterka14Genome Engineering, Discovery Sciences, R&D, AstraZenecaGenome Engineering, Discovery Sciences, R&D, AstraZenecaGenome Engineering, Discovery Sciences, R&D, AstraZenecaTranslational Genomics, Discovery Sciences, R&D, AstraZenecaTranslational Genomics, Discovery Sciences, R&D, AstraZenecaGenome Engineering, Discovery Sciences, R&D, AstraZenecaProtein Science, Structure and Biophysics, Discovery Sciences, R&D, AstraZenecaRNA Therapy, Discovery Sciences, R&D, AstraZenecaRNA Therapy, Discovery Sciences, R&D, AstraZenecaGenome Engineering, Discovery Sciences, R&D, AstraZenecaGenome Engineering, Discovery Sciences, R&D, AstraZenecaData Sciences and Quantitative Biology, Discovery Sciences, R&D, AstraZenecaGenome Engineering, Discovery Sciences, R&D, AstraZenecaGenome Engineering, Discovery Sciences, R&D, AstraZenecaGenome Engineering, Discovery Sciences, R&D, AstraZenecaAbstract Prime editors (PEs) employ reverse transcriptase (RT) to install genomic edits using a template within the prime editing guide RNA (pegRNA). RT creates a 3’ genomic flap containing the intended edit. However, reverse transcription can continue beyond the template, incorporating the pegRNA scaffold sequence into the 3’ flap. These scaffold-derived by-products can be installed alongside the intended edit, reducing prime editing precision. Here, we develop a method that prevents RT from accessing the scaffold, thereby mitigating such by-products. We demonstrate that an internal abasic spacer or 2’-O-methylation within the pegRNAs terminates RT at the end of the template. This prevents scaffold-derived sequences from being incorporated into the target locus. We benchmark these pegRNAs in different cell types and demonstrate that they can be used with processive PEs such as PE6d or PE**. Our findings provide a simple approach to mitigate a common prime editing by-product and improve prime editing precision.https://doi.org/10.1038/s41467-025-58653-1 |
| spellingShingle | Panagiotis Antoniou Louis Dacquay Hanna Mårtensson Katja Madeyski-Bengtson Anna-Lena Loyd Anna Shiriaeva Euan Gordon Salman Mustfa George Thom Pei-Pei Hsieh Saša Šviković Mike Firth Nina Akrap Marcello Maresca Martin Peterka Modified pegRNAs mitigate scaffold-derived prime editing by-products Nature Communications |
| title | Modified pegRNAs mitigate scaffold-derived prime editing by-products |
| title_full | Modified pegRNAs mitigate scaffold-derived prime editing by-products |
| title_fullStr | Modified pegRNAs mitigate scaffold-derived prime editing by-products |
| title_full_unstemmed | Modified pegRNAs mitigate scaffold-derived prime editing by-products |
| title_short | Modified pegRNAs mitigate scaffold-derived prime editing by-products |
| title_sort | modified pegrnas mitigate scaffold derived prime editing by products |
| url | https://doi.org/10.1038/s41467-025-58653-1 |
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