Effects of sgRNAs, Promoters, and Explants on the Gene Editing Efficiency of the CRISPR/Cas9 System in Chinese Kale
The CRISPR/Cas9 system is extensively used for plant gene editing. This study developed an efficient CRISPR/Cas9 system for Chinese kale using multiple sgRNAs and two promoters to create various CRISPR/Cas9 vectors. These vectors targeted <i>BoaZDS</i> and <i>BoaCRTISO</i> in...
| Main Authors: | , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-08-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/24/17/13241 |
| Summary: | The CRISPR/Cas9 system is extensively used for plant gene editing. This study developed an efficient CRISPR/Cas9 system for Chinese kale using multiple sgRNAs and two promoters to create various CRISPR/Cas9 vectors. These vectors targeted <i>BoaZDS</i> and <i>BoaCRTISO</i> in Chinese kale protoplasts and cotyledons. Transient transformation of Chinese kale protoplasts was assessed for editing efficiency at three <i>BoaZDS</i> sites. Notably, sgRNA: Z2 achieved the highest efficiency (90%). Efficiency reached 100% when two sgRNAs targeted <i>BoaZDS</i> with a deletion of a large fragment (576 bp) between them. However, simultaneous targeting of <i>BoaZDS</i> and <i>BoaCRTISO</i> yielded lower efficiency. Transformation of cotyledons led to Chinese kale mutants with albino phenotypes for <i>boazds</i> mutants and orange-mottled phenotypes for <i>boacrtiso</i> mutants. The mutation efficiency of 35S-CRISPR/Cas9 (92.59%) exceeded YAO-CRISPR/Cas9 (70.97%) in protoplasts, and YAO-CRISPR/Cas9 (96.49%) surpassed 35S-CRISPR/Cas9 (58%) in cotyledons. These findings introduce a strategy for enhancing CRISPR/Cas9 editing efficiency in Chinese kale. |
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| ISSN: | 1661-6596 1422-0067 |