Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions
Functional regions that regulate biological phenomena are interspersed throughout eukaryotic genomes. The most definitive approach for identifying such regions is to confirm the phenotype of cells or organisms in which specific regions have been mutated or removed from the genome. This approach is i...
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| Format: | Article |
| Language: | English |
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The Royal Society
2024-04-01
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| Series: | Open Biology |
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| Online Access: | https://royalsocietypublishing.org/doi/10.1098/rsob.240007 |
| _version_ | 1797227216243785728 |
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| author | Kento Morimoto Hayate Suzuki Akihiro Kuno Yoko Daitoku Yoko Tanimoto Kanako Kato Kazuya Murata Fumihiro Sugiyama Seiya Mizuno |
| author_facet | Kento Morimoto Hayate Suzuki Akihiro Kuno Yoko Daitoku Yoko Tanimoto Kanako Kato Kazuya Murata Fumihiro Sugiyama Seiya Mizuno |
| author_sort | Kento Morimoto |
| collection | DOAJ |
| description | Functional regions that regulate biological phenomena are interspersed throughout eukaryotic genomes. The most definitive approach for identifying such regions is to confirm the phenotype of cells or organisms in which specific regions have been mutated or removed from the genome. This approach is invaluable for the functional analysis of genes with a defined functional element, the protein-coding sequence. By contrast, no functional analysis platforms have been established for the study of cis-elements or microRNA cluster regions consisting of multiple microRNAs with functional overlap. Whole-genome mutagenesis approaches, such as via N-ethyl-N-nitrosourea and gene trapping, have greatly contributed to elucidating the function of coding genes. These methods almost never induce deletions of genomic regions or multiple mutations within a narrow region. In other words, cis-elements and microRNA clusters cannot be effectively targeted in such a manner. Herein, we established a novel region-specific random mutagenesis method named CRISPR- and transposase-based regional mutagenesis (CTRL-mutagenesis). We demonstrate that CTRL-mutagenesis randomly induces diverse mutations within target regions in murine embryonic stem cells. Comparative analysis of mutants harbouring subtly different mutations within the same region would facilitate the further study of cis-element and microRNA clusters. |
| first_indexed | 2024-04-24T14:37:16Z |
| format | Article |
| id | doaj.art-45cd721f7963429daf872c1a56ec5bfc |
| institution | Directory Open Access Journal |
| issn | 2046-2441 |
| language | English |
| last_indexed | 2024-04-24T14:37:16Z |
| publishDate | 2024-04-01 |
| publisher | The Royal Society |
| record_format | Article |
| series | Open Biology |
| spelling | doaj.art-45cd721f7963429daf872c1a56ec5bfc2024-04-02T23:05:26ZengThe Royal SocietyOpen Biology2046-24412024-04-0114410.1098/rsob.240007Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regionsKento Morimoto0Hayate Suzuki1Akihiro Kuno2Yoko Daitoku3Yoko Tanimoto4Kanako Kato5Kazuya Murata6Fumihiro Sugiyama7Seiya Mizuno8Doctoral Program in Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, JapanLaboratory Animal Resource Center in Trans-Border Medical Research Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, JapanDepartment of Anatomy and Embryology, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, JapanLaboratory Animal Resource Center in Trans-Border Medical Research Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, JapanLaboratory Animal Resource Center in Trans-Border Medical Research Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, JapanLaboratory Animal Resource Center in Trans-Border Medical Research Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, JapanLaboratory Animal Resource Center in Trans-Border Medical Research Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, JapanLaboratory Animal Resource Center in Trans-Border Medical Research Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, JapanLaboratory Animal Resource Center in Trans-Border Medical Research Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, JapanFunctional regions that regulate biological phenomena are interspersed throughout eukaryotic genomes. The most definitive approach for identifying such regions is to confirm the phenotype of cells or organisms in which specific regions have been mutated or removed from the genome. This approach is invaluable for the functional analysis of genes with a defined functional element, the protein-coding sequence. By contrast, no functional analysis platforms have been established for the study of cis-elements or microRNA cluster regions consisting of multiple microRNAs with functional overlap. Whole-genome mutagenesis approaches, such as via N-ethyl-N-nitrosourea and gene trapping, have greatly contributed to elucidating the function of coding genes. These methods almost never induce deletions of genomic regions or multiple mutations within a narrow region. In other words, cis-elements and microRNA clusters cannot be effectively targeted in such a manner. Herein, we established a novel region-specific random mutagenesis method named CRISPR- and transposase-based regional mutagenesis (CTRL-mutagenesis). We demonstrate that CTRL-mutagenesis randomly induces diverse mutations within target regions in murine embryonic stem cells. Comparative analysis of mutants harbouring subtly different mutations within the same region would facilitate the further study of cis-element and microRNA clusters.https://royalsocietypublishing.org/doi/10.1098/rsob.240007region-specific random mutagenesisCRISPR-Cas9PiggyBacnon-coding regionmicroRNA cluster |
| spellingShingle | Kento Morimoto Hayate Suzuki Akihiro Kuno Yoko Daitoku Yoko Tanimoto Kanako Kato Kazuya Murata Fumihiro Sugiyama Seiya Mizuno Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions Open Biology region-specific random mutagenesis CRISPR-Cas9 PiggyBac non-coding region microRNA cluster |
| title | Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions |
| title_full | Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions |
| title_fullStr | Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions |
| title_full_unstemmed | Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions |
| title_short | Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions |
| title_sort | regional random mutagenesis driven by multiple sgrnas and diverse on target genome editing events to identify functionally important elements in non coding regions |
| topic | region-specific random mutagenesis CRISPR-Cas9 PiggyBac non-coding region microRNA cluster |
| url | https://royalsocietypublishing.org/doi/10.1098/rsob.240007 |
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