دورية أكاديمية
Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis
| العنوان: | Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis |
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| المؤلفون: | Susan Schröpfer, Henryk Flachowsky |
| المصدر: | International Journal of Molecular Sciences, Vol 22, Iss 22, p 12611 (2021) |
| بيانات النشر: | MDPI AG, 2021. |
| سنة النشر: | 2021 |
| المجموعة: | LCC:Biology (General) LCC:Chemistry |
| مصطلحات موضوعية: | Malus domestica, Cpf1, MdPDS, fragment length analysis, amplicon deep sequencing, deletion, Biology (General), QH301-705.5, Chemistry, QD1-999 |
| الوصف: | The use of the novel CRISPR/Cas12a system is advantageous, as it expands the possibilities for genome editing (GE) applications due to its different features compared to the commonly used CRISPR/Cas9 system. In this work, the CRISPR/Cas12a system was applied for the first time to apple to investigate its general usability for GE applications. Efficient guide RNAs targeting different exons of the endogenous reporter gene MdPDS, whose disruption leads to the albino phenotype, were pre-selected by in vitro cleavage assays. A construct was transferred to apple encoding for a CRISPR/Cas12a system that simultaneously targets two loci in MdPDS. Using fluorescent PCR capillary electrophoresis and amplicon deep sequencing, all identified GE events of regenerated albino shoots were characterized as deletions. Large deletions between the two neighboring target sites were not observed. Furthermore, a chimeric composition of regenerates and shoots that exhibited multiple GE events was observed frequently. By comparing both analytical methods, it was shown that fluorescent PCR capillary gel electrophoresis is a sensitive high-throughput genotyping method that allows accurate predictions of the size and proportion of indel mutations for multiple loci simultaneously. Especially for species exhibiting high frequencies of chimerism, it can be recommended as a cost-effective method for efficient selection of homohistont GE lines. |
| نوع الوثيقة: | article |
| وصف الملف: | electronic resource |
| اللغة: | English |
| تدمد: | 1422-0067 1661-6596 |
| Relation: | https://www.mdpi.com/1422-0067/22/22/12611; https://doaj.org/toc/1661-6596; https://doaj.org/toc/1422-0067 |
| DOI: | 10.3390/ijms222212611 |
| URL الوصول: | https://doaj.org/article/9495efe019234640869374ef065a493d |
| رقم الأكسشن: | edsdoj.9495efe019234640869374ef065a493d |
| قاعدة البيانات: | Directory of Open Access Journals |
Full Text Finder | تدمد: | 14220067 16616596 |
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| DOI: | 10.3390/ijms222212611 |