CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations
Abstract Cell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that ena...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2021-12-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-021-02986-6 |
| _version_ | 1819009365107015680 |
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| author | Tsuyoshi Fukushima Yosuke Tanaka Keito Adachi Nanami Masuyama Akiho Tsuchiya Shuhei Asada Soh Ishiguro Hideto Mori Motoaki Seki Nozomu Yachie Susumu Goyama Toshio Kitamura |
| author_facet | Tsuyoshi Fukushima Yosuke Tanaka Keito Adachi Nanami Masuyama Akiho Tsuchiya Shuhei Asada Soh Ishiguro Hideto Mori Motoaki Seki Nozomu Yachie Susumu Goyama Toshio Kitamura |
| author_sort | Tsuyoshi Fukushima |
| collection | DOAJ |
| description | Abstract Cell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA activation in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, thymidine (T)-to-cytosine (C) substitutions in the scaffold region of the sgRNA or the TATA box-containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, leading to activation of the next sgRNA. These reactions can occur multiple times, resulting in cellular chain reactions. As a proof of concept, we established a chain reaction by repairing sgRNA scaffold mutations in 293 T cells. Importantly, the results obtained in yeast or in vitro did not match those obtained in mammalian cells, suggesting that in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research. |
| first_indexed | 2024-12-21T00:55:12Z |
| format | Article |
| id | doaj.art-e60e48ea503548f2a78b8b97e2db5163 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-21T00:55:12Z |
| publishDate | 2021-12-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-e60e48ea503548f2a78b8b97e2db51632022-12-21T19:21:19ZengNature PortfolioScientific Reports2045-23222021-12-0111111210.1038/s41598-021-02986-6CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutationsTsuyoshi Fukushima0Yosuke Tanaka1Keito Adachi2Nanami Masuyama3Akiho Tsuchiya4Shuhei Asada5Soh Ishiguro6Hideto Mori7Motoaki Seki8Nozomu Yachie9Susumu Goyama10Toshio Kitamura11Division of Cellular Therapy, The Institute of Medical Science, The University of TokyoDivision of Cellular Therapy, The Institute of Medical Science, The University of TokyoDivision of Cellular Therapy, The Institute of Medical Science, The University of TokyoResearch Center for Advanced Science and Technology, The University of TokyoDivision of Cellular Therapy, The Institute of Medical Science, The University of TokyoDivision of Cellular Therapy, The Institute of Medical Science, The University of TokyoResearch Center for Advanced Science and Technology, The University of TokyoResearch Center for Advanced Science and Technology, The University of TokyoResearch Center for Advanced Science and Technology, The University of TokyoResearch Center for Advanced Science and Technology, The University of TokyoDivision of Molecular Oncology, Graduate School of Frontier Sciences, The Institute of Medical Science, The University of TokyoDivision of Cellular Therapy, The Institute of Medical Science, The University of TokyoAbstract Cell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA activation in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, thymidine (T)-to-cytosine (C) substitutions in the scaffold region of the sgRNA or the TATA box-containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, leading to activation of the next sgRNA. These reactions can occur multiple times, resulting in cellular chain reactions. As a proof of concept, we established a chain reaction by repairing sgRNA scaffold mutations in 293 T cells. Importantly, the results obtained in yeast or in vitro did not match those obtained in mammalian cells, suggesting that in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.https://doi.org/10.1038/s41598-021-02986-6 |
| spellingShingle | Tsuyoshi Fukushima Yosuke Tanaka Keito Adachi Nanami Masuyama Akiho Tsuchiya Shuhei Asada Soh Ishiguro Hideto Mori Motoaki Seki Nozomu Yachie Susumu Goyama Toshio Kitamura CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations Scientific Reports |
| title | CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations |
| title_full | CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations |
| title_fullStr | CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations |
| title_full_unstemmed | CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations |
| title_short | CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations |
| title_sort | crispr cas9 mediated base editing enables a chain reaction through sequential repair of sgrna scaffold mutations |
| url | https://doi.org/10.1038/s41598-021-02986-6 |
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