A molecular proximity sensor based on an engineered, dual-component guide RNA
One of the goals of synthetic biology is to enable the design of arbitrary molecular circuits with programmable inputs and outputs. Such circuits bridge the properties of electronic and natural circuits, processing information in a predictable manner within living cells. Genome editing is a potentia...
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2025-02-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/98110 |
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| author | Junhong Choi Wei Chen Hanna Liao Xiaoyi Li Jay Shendure |
| author_facet | Junhong Choi Wei Chen Hanna Liao Xiaoyi Li Jay Shendure |
| author_sort | Junhong Choi |
| collection | DOAJ |
| description | One of the goals of synthetic biology is to enable the design of arbitrary molecular circuits with programmable inputs and outputs. Such circuits bridge the properties of electronic and natural circuits, processing information in a predictable manner within living cells. Genome editing is a potentially powerful component of synthetic molecular circuits, whether for modulating the expression of a target gene or for stably recording information to genomic DNA. However, programming molecular events such as protein-protein interactions or induced proximity as triggers for genome editing remains challenging. Here, we demonstrate a strategy termed ‘P3 editing’, which links protein-protein proximity to the formation of a functional CRISPR-Cas9 dual-component guide RNA. By engineering the crRNA:tracrRNA interaction, we demonstrate that various known protein-protein interactions, as well as the chemically induced dimerization of protein domains, can be used to activate prime editing or base editing in human cells. Additionally, we explore how P3 editing can incorporate outputs from ADAR-based RNA sensors, potentially allowing specific RNAs to induce specific genome edits within a larger circuit. Our strategy enhances the controllability of CRISPR-based genome editing, facilitating its use in synthetic molecular circuits deployed in living cells. |
| format | Article |
| id | doaj-art-0813d6fe14ea444fb76ed6130ec237c5 |
| institution | DOAJ |
| issn | 2050-084X |
| language | English |
| publishDate | 2025-02-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj-art-0813d6fe14ea444fb76ed6130ec237c52025-08-20T02:48:06ZengeLife Sciences Publications LtdeLife2050-084X2025-02-011310.7554/eLife.98110A molecular proximity sensor based on an engineered, dual-component guide RNAJunhong Choi0https://orcid.org/0000-0001-9291-5977Wei Chen1Hanna Liao2Xiaoyi Li3Jay Shendure4https://orcid.org/0000-0002-1516-1865Department of Genome Sciences, University of Washington, Seattle, United States; Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, United StatesDepartment of Genome Sciences, University of Washington, Seattle, United States; Institute for Protein Design, University of Washington, Seattle, United StatesDepartment of Genome Sciences, University of Washington, Seattle, United States; Molecular and Cellular Biology Program, University of Washington, Seattle, United StatesDepartment of Genome Sciences, University of Washington, Seattle, United StatesDepartment of Genome Sciences, University of Washington, Seattle, United States; Howard Hughes Medical Institute, Seattle, United States; Brotman Baty Institute for Precision Medicine, Seattle, United States; Allen Discovery Center for Cell Lineage Tracing, Seattle, United States; Seattle Hub for Synthetic Biology, Seattle, United StatesOne of the goals of synthetic biology is to enable the design of arbitrary molecular circuits with programmable inputs and outputs. Such circuits bridge the properties of electronic and natural circuits, processing information in a predictable manner within living cells. Genome editing is a potentially powerful component of synthetic molecular circuits, whether for modulating the expression of a target gene or for stably recording information to genomic DNA. However, programming molecular events such as protein-protein interactions or induced proximity as triggers for genome editing remains challenging. Here, we demonstrate a strategy termed ‘P3 editing’, which links protein-protein proximity to the formation of a functional CRISPR-Cas9 dual-component guide RNA. By engineering the crRNA:tracrRNA interaction, we demonstrate that various known protein-protein interactions, as well as the chemically induced dimerization of protein domains, can be used to activate prime editing or base editing in human cells. Additionally, we explore how P3 editing can incorporate outputs from ADAR-based RNA sensors, potentially allowing specific RNAs to induce specific genome edits within a larger circuit. Our strategy enhances the controllability of CRISPR-based genome editing, facilitating its use in synthetic molecular circuits deployed in living cells.https://elifesciences.org/articles/98110synthetic biologygenome editingmolecular recordingCRISPR-Casprotein-protein interaction |
| spellingShingle | Junhong Choi Wei Chen Hanna Liao Xiaoyi Li Jay Shendure A molecular proximity sensor based on an engineered, dual-component guide RNA eLife synthetic biology genome editing molecular recording CRISPR-Cas protein-protein interaction |
| title | A molecular proximity sensor based on an engineered, dual-component guide RNA |
| title_full | A molecular proximity sensor based on an engineered, dual-component guide RNA |
| title_fullStr | A molecular proximity sensor based on an engineered, dual-component guide RNA |
| title_full_unstemmed | A molecular proximity sensor based on an engineered, dual-component guide RNA |
| title_short | A molecular proximity sensor based on an engineered, dual-component guide RNA |
| title_sort | molecular proximity sensor based on an engineered dual component guide rna |
| topic | synthetic biology genome editing molecular recording CRISPR-Cas protein-protein interaction |
| url | https://elifesciences.org/articles/98110 |
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