CRISPR-Combo–mediated orthogonal genome editing and transcriptional activation for plant breeding
CRISPR-Combo–mediated orthogonal genome editing and transcriptional activation for plant breeding
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DOI：
10.1038/s41596-023-00823-w
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发表时间：
2023-04
期刊：
影响因子：
14.8
通讯作者：
Changtian Pan;Yiping Qi
中科院分区：
文献类型：
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作者：
Changtian Pan;Yiping Qi
CRISPR-Cas核酸酶系统、碱基编辑器和CRISPR激活极大地推进了植物基因组工程。然而，用于多重正交基因组编辑和转录调控的组合方法先前未在植物中开发。我们最近建立了一个基于单一Cas9蛋白的CRISPR-Combo平台，通过工程化单向导RNA（sgRNA）结构，实现了植物中高效的多重正交基因组编辑（双链断裂介导的基因组编辑或碱基编辑）和转录激活。在这里，我们提供了构建CRISPR-Combo系统的分步说明，以快速培育无转基因、基因组编辑的拟南芥植物，并以无突变的方式用更多可遗传的靶向突变增强水稻再生。我们还为设计高效的sgRNA、农杆菌介导的拟南芥和水稻的转化、无外源植物激素的水稻再生、基因编辑评价和视觉识别具有高编辑活性的无转基因拟南芥植株提供指导。通过使用该方案，建立CRISPR-Combo系统需要约2周，获得无转基因组编辑的水稻植物需要4个月，通过无组织培养获得富含可遗传靶向突变的水稻植物需要4个月。
CRISPR–Cas nuclease systems, base editors, and CRISPR activation have greatly advanced plant genome engineering. However, the combinatorial approaches for multiplexed orthogonal genome editing and transcriptional regulation were previously unexploited in plants. We have recently established a single Cas9 protein-based CRISPR-Combo platform, enabling efficient multiplexed orthogonal genome editing (double-strand break–mediated genome editing or base editing) and transcriptional activation in plants via engineering the single guide RNA (sgRNA) structure. Here, we provide step-by-step instructions for constructing CRISPR-Combo systems for speed breeding of transgene-free, genome-editedArabidopsisplants and enhancing rice regeneration with more heritable targeted mutations in a hormone-free manner. We also provide guidance on designing efficient sgRNA,Agrobacterium-mediated transformation ofArabidopsisand rice, rice regeneration without exogenous plant hormones, gene editing evaluation and visual identification of transgene-freeArabidopsisplants with high editing activity. With the use of this protocol, it takes ~2 weeks to establish the CRISPR-Combo systems, 4 months to obtain transgene-free genome-editedArabidopsisplants and 4 months to obtain rice plants with enrichment of heritable targeted mutations by hormone-free tissue culture.