CRISPR-finder: A high throughput and cost-effective method to identify successfully edited Arabidopsis thaliana individuals
Genome editing with the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated protein) system allows mutagenesis of a targeted region of the genome using a Cas endonuclease and an artificial guide RNA. Both because of variable efficiency with which such mutations ar...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Cambridge University Press
2021-01-01
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| Series: | Quantitative Plant Biology |
| Subjects: | |
| Online Access: | https://www.cambridge.org/core/product/identifier/S2632882820000065/type/journal_article |
| _version_ | 1811155767854628864 |
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| author | Efthymia Symeonidi Julian Regalado Rebecca Schwab Detlef Weigel |
| author_facet | Efthymia Symeonidi Julian Regalado Rebecca Schwab Detlef Weigel |
| author_sort | Efthymia Symeonidi |
| collection | DOAJ |
| description | Genome editing with the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated protein) system allows mutagenesis of a targeted region of the genome using a Cas endonuclease and an artificial guide RNA. Both because of variable efficiency with which such mutations arise and because the repair process produces a spectrum of mutations, one needs to ascertain the genome sequence at the targeted locus for many individuals that have been subjected to mutagenesis. We provide a complete protocol for the generation of amplicons up until the identification of the exact mutations in the targeted region. CRISPR-finder can be used to process thousands of individuals in a single sequencing run. We successfully identified an ISOCHORISMATE SYNTHASE 1 mutant line in which the production of salicylic acid was impaired compared to the wild type, as expected. These features establish CRISPR-finder as a high-throughput, cost-effective and efficient genotyping method of individuals whose genomes have been targeted using the CRISPR/Cas9 system. |
| first_indexed | 2024-04-10T04:39:05Z |
| format | Article |
| id | doaj.art-dbbca92e5ae84bc6aace7d7c67d8ac5e |
| institution | Directory Open Access Journal |
| issn | 2632-8828 |
| language | English |
| last_indexed | 2024-04-10T04:39:05Z |
| publishDate | 2021-01-01 |
| publisher | Cambridge University Press |
| record_format | Article |
| series | Quantitative Plant Biology |
| spelling | doaj.art-dbbca92e5ae84bc6aace7d7c67d8ac5e2023-03-09T12:43:33ZengCambridge University PressQuantitative Plant Biology2632-88282021-01-01210.1017/qpb.2020.6CRISPR-finder: A high throughput and cost-effective method to identify successfully edited Arabidopsis thaliana individualsEfthymia Symeonidi0https://orcid.org/0000-0002-4412-4596Julian Regalado1Rebecca Schwab2Detlef Weigel3https://orcid.org/0000-0002-2114-7963Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, GermanyDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, GermanyDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, GermanyDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, GermanyGenome editing with the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated protein) system allows mutagenesis of a targeted region of the genome using a Cas endonuclease and an artificial guide RNA. Both because of variable efficiency with which such mutations arise and because the repair process produces a spectrum of mutations, one needs to ascertain the genome sequence at the targeted locus for many individuals that have been subjected to mutagenesis. We provide a complete protocol for the generation of amplicons up until the identification of the exact mutations in the targeted region. CRISPR-finder can be used to process thousands of individuals in a single sequencing run. We successfully identified an ISOCHORISMATE SYNTHASE 1 mutant line in which the production of salicylic acid was impaired compared to the wild type, as expected. These features establish CRISPR-finder as a high-throughput, cost-effective and efficient genotyping method of individuals whose genomes have been targeted using the CRISPR/Cas9 system.https://www.cambridge.org/core/product/identifier/S2632882820000065/type/journal_articleamplicon sequencingCRISPR/Cas9ICS1salicylic acid |
| spellingShingle | Efthymia Symeonidi Julian Regalado Rebecca Schwab Detlef Weigel CRISPR-finder: A high throughput and cost-effective method to identify successfully edited Arabidopsis thaliana individuals Quantitative Plant Biology amplicon sequencing CRISPR/Cas9 ICS1 salicylic acid |
| title | CRISPR-finder: A high throughput and cost-effective method to identify successfully edited Arabidopsis thaliana individuals |
| title_full | CRISPR-finder: A high throughput and cost-effective method to identify successfully edited Arabidopsis thaliana individuals |
| title_fullStr | CRISPR-finder: A high throughput and cost-effective method to identify successfully edited Arabidopsis thaliana individuals |
| title_full_unstemmed | CRISPR-finder: A high throughput and cost-effective method to identify successfully edited Arabidopsis thaliana individuals |
| title_short | CRISPR-finder: A high throughput and cost-effective method to identify successfully edited Arabidopsis thaliana individuals |
| title_sort | crispr finder a high throughput and cost effective method to identify successfully edited arabidopsis thaliana individuals |
| topic | amplicon sequencing CRISPR/Cas9 ICS1 salicylic acid |
| url | https://www.cambridge.org/core/product/identifier/S2632882820000065/type/journal_article |
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