Gene duplication followed by sequence diversification is a key driver of innovation in genome evolution. To mimic this process in genome engineering, a method for region-restricted mutagenesis is needed to selectively mutate one copy of a duplicated gene. Notably, regions flanking a double-strand break (DSB) become hypersensitive to mutagens due to end resection, which converts them into single-stranded DNA (ssDNA). Blocking end resection could, therefore, confine hypermutation to a limited region. To achieve this, we investigated a catalytically inactive variant of Streptococcus pyogenes Cas9 (dSpCas9) and demonstrated its ability to attenuate end resection in the budding yeast Saccharomyces cerevisiae using ssDNA-specific quantitative PCR, live-cell imaging, and Southern blot analysis. By leveraging the bisulfite sensitivity of ssDNA, we further validated the concept of DSB-coupled, dSpCas9-mediated region-restricted mutagenesis. We anticipate that dSpCas9-mediated modulation of end resection at induced DSB sites will have valuable applications in both genome engineering and mechanistic studies.
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| Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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| Site | Modification | Modifier | Source | Reference |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Increase the total number of rows showing on this page by using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small "i" buttons located within a cell for an annotation to view further details about experiment type and any other genes involved in the interaction.
| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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| Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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| Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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