Parallel genetics of regulatory sequences using scalable genome editing in vivo
Summary: How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. Regulatory elements must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regu...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2021-04-01
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| Series: | Cell Reports |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2211124721003028 |
| _version_ | 1818971111969259520 |
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| author | Jonathan J. Froehlich Bora Uyar Margareta Herzog Kathrin Theil Petar Glažar Altuna Akalin Nikolaus Rajewsky |
| author_facet | Jonathan J. Froehlich Bora Uyar Margareta Herzog Kathrin Theil Petar Glažar Altuna Akalin Nikolaus Rajewsky |
| author_sort | Jonathan J. Froehlich |
| collection | DOAJ |
| description | Summary: How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. Regulatory elements must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3′ UTRs of 16 genes in C. elegans. Our software crispr-DART analyzes indel mutations in targeted DNA sequencing. We quantify the impact of mutations on expression and fitness by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3′ UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression independently of each other. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of regulatory sequences directly in animals. |
| first_indexed | 2024-12-20T14:47:11Z |
| format | Article |
| id | doaj.art-ce5cdaf5f7314b4398c31f465e7e436e |
| institution | Directory Open Access Journal |
| issn | 2211-1247 |
| language | English |
| last_indexed | 2024-12-20T14:47:11Z |
| publishDate | 2021-04-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cell Reports |
| spelling | doaj.art-ce5cdaf5f7314b4398c31f465e7e436e2022-12-21T19:37:05ZengElsevierCell Reports2211-12472021-04-01352108988Parallel genetics of regulatory sequences using scalable genome editing in vivoJonathan J. Froehlich0Bora Uyar1Margareta Herzog2Kathrin Theil3Petar Glažar4Altuna Akalin5Nikolaus Rajewsky6Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, GermanyBioinformatics and Omics Data Science Platform, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, GermanySystems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, GermanySystems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, GermanySystems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, GermanyBioinformatics and Omics Data Science Platform, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, GermanySystems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str. 28, 10115 Berlin, Germany; Corresponding authorSummary: How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. Regulatory elements must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3′ UTRs of 16 genes in C. elegans. Our software crispr-DART analyzes indel mutations in targeted DNA sequencing. We quantify the impact of mutations on expression and fitness by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3′ UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression independently of each other. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of regulatory sequences directly in animals.http://www.sciencedirect.com/science/article/pii/S2211124721003028gene regulationgene-regulatory elementsanimal phenotypeCaenorhabditis elegansin vivoCRISPR-Cas9 |
| spellingShingle | Jonathan J. Froehlich Bora Uyar Margareta Herzog Kathrin Theil Petar Glažar Altuna Akalin Nikolaus Rajewsky Parallel genetics of regulatory sequences using scalable genome editing in vivo Cell Reports gene regulation gene-regulatory elements animal phenotype Caenorhabditis elegans in vivo CRISPR-Cas9 |
| title | Parallel genetics of regulatory sequences using scalable genome editing in vivo |
| title_full | Parallel genetics of regulatory sequences using scalable genome editing in vivo |
| title_fullStr | Parallel genetics of regulatory sequences using scalable genome editing in vivo |
| title_full_unstemmed | Parallel genetics of regulatory sequences using scalable genome editing in vivo |
| title_short | Parallel genetics of regulatory sequences using scalable genome editing in vivo |
| title_sort | parallel genetics of regulatory sequences using scalable genome editing in vivo |
| topic | gene regulation gene-regulatory elements animal phenotype Caenorhabditis elegans in vivo CRISPR-Cas9 |
| url | http://www.sciencedirect.com/science/article/pii/S2211124721003028 |
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