Robust prediction of synthetic gRNA activity and cryptic DNA repair by disentangling cellular CRISPR cleavage outcomes
Abstract The ability to robustly predict guide RNA (gRNA) activity is a long-standing goal for CRISPR applications, as it would reduce the need to pre-screen gRNAs. Quantification of formation of short insertions and deletions (indels) after DNA cleavage by transcribed gRNAs has been typically used...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-05-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-59947-0 |
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| author | Stephan Riesenberg Philipp Kanis Rosa Karlic Tomislav Maricic |
| author_facet | Stephan Riesenberg Philipp Kanis Rosa Karlic Tomislav Maricic |
| author_sort | Stephan Riesenberg |
| collection | DOAJ |
| description | Abstract The ability to robustly predict guide RNA (gRNA) activity is a long-standing goal for CRISPR applications, as it would reduce the need to pre-screen gRNAs. Quantification of formation of short insertions and deletions (indels) after DNA cleavage by transcribed gRNAs has been typically used to measure and predict gRNA activity. We evaluate the effect of chemically synthesized Cas9 gRNAs on different cellular DNA cleavage outcomes and find that the activity of different gRNAs is largely similar and often underestimated when only indels are scored. We provide a simple linear model that reliably predicts synthetic gRNA activity across cell lines, robustly identifies inefficient gRNAs across different published datasets, and is easily accessible via online genome browser tracks. In addition, we develop a homology-directed repair efficiency prediction tool and show that unintended large-scale repair events are common for Cas9 but not for Cas12a, which may be relevant for safety in gene therapy applications. |
| format | Article |
| id | doaj-art-55ea47c5af2d4417962e625b849cfa2e |
| institution | OA Journals |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-55ea47c5af2d4417962e625b849cfa2e2025-08-20T02:29:51ZengNature PortfolioNature Communications2041-17232025-05-0116111410.1038/s41467-025-59947-0Robust prediction of synthetic gRNA activity and cryptic DNA repair by disentangling cellular CRISPR cleavage outcomesStephan Riesenberg0Philipp Kanis1Rosa Karlic2Tomislav Maricic3Department of Evolutionary Genetics, Max Planck Institute for Evolutionary AnthropologyDepartment of Evolutionary Genetics, Max Planck Institute for Evolutionary AnthropologyBioinformatics Group, Division of Molecular Biology, Department of Biology, University of ZagrebDepartment of Evolutionary Genetics, Max Planck Institute for Evolutionary AnthropologyAbstract The ability to robustly predict guide RNA (gRNA) activity is a long-standing goal for CRISPR applications, as it would reduce the need to pre-screen gRNAs. Quantification of formation of short insertions and deletions (indels) after DNA cleavage by transcribed gRNAs has been typically used to measure and predict gRNA activity. We evaluate the effect of chemically synthesized Cas9 gRNAs on different cellular DNA cleavage outcomes and find that the activity of different gRNAs is largely similar and often underestimated when only indels are scored. We provide a simple linear model that reliably predicts synthetic gRNA activity across cell lines, robustly identifies inefficient gRNAs across different published datasets, and is easily accessible via online genome browser tracks. In addition, we develop a homology-directed repair efficiency prediction tool and show that unintended large-scale repair events are common for Cas9 but not for Cas12a, which may be relevant for safety in gene therapy applications.https://doi.org/10.1038/s41467-025-59947-0 |
| spellingShingle | Stephan Riesenberg Philipp Kanis Rosa Karlic Tomislav Maricic Robust prediction of synthetic gRNA activity and cryptic DNA repair by disentangling cellular CRISPR cleavage outcomes Nature Communications |
| title | Robust prediction of synthetic gRNA activity and cryptic DNA repair by disentangling cellular CRISPR cleavage outcomes |
| title_full | Robust prediction of synthetic gRNA activity and cryptic DNA repair by disentangling cellular CRISPR cleavage outcomes |
| title_fullStr | Robust prediction of synthetic gRNA activity and cryptic DNA repair by disentangling cellular CRISPR cleavage outcomes |
| title_full_unstemmed | Robust prediction of synthetic gRNA activity and cryptic DNA repair by disentangling cellular CRISPR cleavage outcomes |
| title_short | Robust prediction of synthetic gRNA activity and cryptic DNA repair by disentangling cellular CRISPR cleavage outcomes |
| title_sort | robust prediction of synthetic grna activity and cryptic dna repair by disentangling cellular crispr cleavage outcomes |
| url | https://doi.org/10.1038/s41467-025-59947-0 |
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