期刊
FRONTIERS IN PLANT SCIENCE
卷 13, 期 -, 页码 -出版社
FRONTIERS MEDIA SA
DOI: 10.3389/fpls.2022.1010030
关键词
genome editing; plant biotechnology; protoplast; sgRNA scaffold; stomatal development; targeted mutagenesis; CRISPR; Cas9; Zea mays
资金
- Investissement d'Avenir program of the French National Agency of Research for the project GENIUS [2018/0480]
- CIFRE fellowship of the ANRT
- [ANR-11-BTBR-0001_GENIUS]
In this study, a maize protoplast system coupled with NGS analysis and a novel bioinformatics pipeline was established to evaluate the efficiency of CRISPR/Cas9 tools. The results showed a correlation between mutation types observed in protoplasts and those obtained after stable transformation. Additionally, factors influencing genome editing efficiency were identified, and phenotypic analysis was conducted on base-edited maize plants.
IntroductionDespite its rapid worldwide adoption as an efficient mutagenesis tool, plant genome editing remains a labor-intensive process requiring often several months of in vitro culture to obtain mutant plantlets. To avoid a waste in time and money and to test, in only a few days, the efficiency of molecular constructs or novel Cas9 variants (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9) prior to stable transformation, rapid analysis tools are helpful. MethodsTo this end, a streamlined maize protoplast system for transient expression of CRISPR/Cas9 tools coupled to NGS (next generation sequencing) analysis and a novel bioinformatics pipeline was established. Results and discussionMutation types found with high frequency in maize leaf protoplasts had a trend to be the ones observed after stable transformation of immature maize embryos. The protoplast system also allowed to conclude that modifications of the sgRNA (single guide RNA) scaffold leave little room for improvement, that relaxed PAM (protospacer adjacent motif) sites increase the choice of target sites for genome editing, albeit with decreased frequency, and that efficient base editing in maize could be achieved for certain but not all target sites. Phenotypic analysis of base edited mutant maize plants demonstrated that the introduction of a stop codon but not the mutation of a serine predicted to be phosphorylated in the bHLH (basic helix loop helix) transcription factor ZmICEa (INDUCER OF CBF EXPRESSIONa) caused abnormal stomata, pale leaves and eventual plant death two months after sowing.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据