Protocol for generating splice isoform-specific mouse mutants using CRISPR-Cas9 and a minigene splicing reporter
Summary: Here, we present a protocol to alter the production of alternatively spliced mRNA variants, without affecting the overall gene expression, through CRISPR-Cas9-engineered genomic mutations in mice. We describe steps for designing guide RNA to direct Cas9 endonuclease to consensus splice site...
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| Language: | English |
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Elsevier
2025-03-01
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| Series: | STAR Protocols |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166724007081 |
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| author | Yudong Teng Kelsey Arbogast Harald Junge Zhe Chen |
| author_facet | Yudong Teng Kelsey Arbogast Harald Junge Zhe Chen |
| author_sort | Yudong Teng |
| collection | DOAJ |
| description | Summary: Here, we present a protocol to alter the production of alternatively spliced mRNA variants, without affecting the overall gene expression, through CRISPR-Cas9-engineered genomic mutations in mice. We describe steps for designing guide RNA to direct Cas9 endonuclease to consensus splice sites, producing transgenic mice through pronuclear injection, and screening for desired mutations in cultured mammalian cells using a minigene splicing reporter. Splice isoform-specific mouse mutants provide valuable tools for genetic analyses beyond loss-of-function and transgenic alleles.For complete details on the use and execution of this protocol, please refer to Dailey-Krempel et al.1 and Johnson et al.2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-04d76d5e199e4f4db05eba7e2e044ffc |
| institution | Kabale University |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-04d76d5e199e4f4db05eba7e2e044ffc2025-01-05T04:28:40ZengElsevierSTAR Protocols2666-16672025-03-0161103543Protocol for generating splice isoform-specific mouse mutants using CRISPR-Cas9 and a minigene splicing reporterYudong Teng0Kelsey Arbogast1Harald Junge2Zhe Chen3The Genetically Engineered Murine Models Core, Department of Immunology & Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USAColorado Center for Personalized Medicine Biobank, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USADepartment of Ophthalmology and Visual Neuroscience, University of Minnesota Medical School, Minneapolis, MN 55455, USADepartment of Neuroscience, University of Minnesota Medical School, Minneapolis, MN 55455, USA; Corresponding authorSummary: Here, we present a protocol to alter the production of alternatively spliced mRNA variants, without affecting the overall gene expression, through CRISPR-Cas9-engineered genomic mutations in mice. We describe steps for designing guide RNA to direct Cas9 endonuclease to consensus splice sites, producing transgenic mice through pronuclear injection, and screening for desired mutations in cultured mammalian cells using a minigene splicing reporter. Splice isoform-specific mouse mutants provide valuable tools for genetic analyses beyond loss-of-function and transgenic alleles.For complete details on the use and execution of this protocol, please refer to Dailey-Krempel et al.1 and Johnson et al.2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166724007081CRISPRDevelopmental biologyModel OrganismsMolecular BiologyNeuroscience |
| spellingShingle | Yudong Teng Kelsey Arbogast Harald Junge Zhe Chen Protocol for generating splice isoform-specific mouse mutants using CRISPR-Cas9 and a minigene splicing reporter STAR Protocols CRISPR Developmental biology Model Organisms Molecular Biology Neuroscience |
| title | Protocol for generating splice isoform-specific mouse mutants using CRISPR-Cas9 and a minigene splicing reporter |
| title_full | Protocol for generating splice isoform-specific mouse mutants using CRISPR-Cas9 and a minigene splicing reporter |
| title_fullStr | Protocol for generating splice isoform-specific mouse mutants using CRISPR-Cas9 and a minigene splicing reporter |
| title_full_unstemmed | Protocol for generating splice isoform-specific mouse mutants using CRISPR-Cas9 and a minigene splicing reporter |
| title_short | Protocol for generating splice isoform-specific mouse mutants using CRISPR-Cas9 and a minigene splicing reporter |
| title_sort | protocol for generating splice isoform specific mouse mutants using crispr cas9 and a minigene splicing reporter |
| topic | CRISPR Developmental biology Model Organisms Molecular Biology Neuroscience |
| url | http://www.sciencedirect.com/science/article/pii/S2666166724007081 |
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