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在葡萄藤中使用CRISPR/LbCas12a系统进行高效的基因组编辑
Chong Ren1,2,3, Elias Kirabi Gathunga1,2,3, Xue Li1,2,3
1State Key Laboratory of Plant Diversity and Specialty Crops, Beijing Key Laboratory of Grape Sciences and Enology, Institute of Botany, the Chinese Academy of Sciences, Beijing, 100093, PR China.
Molecular horticulture
|October 18, 2023
概括
现在,CRISPR/Cas12a系统,特别是LbCas12a,已经有效地用于葡萄藤基因组工程. 该系统成功诱导了目标基因的突变,热处理提高了效率,截断的crRNA显示出精确基因编辑的前景.
科学领域:
- 植物生物技术 植物生物技术
- 分子生物学分子生物学
- 遗传学 遗传学 是一个
背景情况:
- 集群定期间隔的短时间Palindromic重复 (CRISPR) /Cas12a系统在各种植物中提供了强大的基因组工程能力.
- 葡萄藤 (Vitis vinifera) 尚未受益于CRISPR/Cas12a用于基因改造的应用.
研究的目的:
- 开发和验证CRISPR/Cas12a系统,特别是Lachnospiraceae细菌ND2006 Cas12a (LbCas12a),用于葡萄藤细胞的向突变.
- 调查基因淘汰对黄积累的影响,并通过环境和分子修改优化编辑效率.
主要方法:
- 采用CRISPR/LbCas12a来准葡萄藤41B细胞中的塑性单糖转运体1 (TMT1) 和二黄-4-减少酶1 (DFR1) 基因.
- 诱导了变异,并评估了热处理 (34°C) 对编辑效率的影响.
- 分析了crRNA序列长度和截断crRNAs (trucrRNAs) 对基因组编辑结果的影响.
主要成果:
- 通过敲除DFR1基因,在葡萄41B细胞中成功实现了向性突变发生,从而导致改变了黄类积累.
- 热处理显著提高了TMT1的CRISPR/LbCas12a编辑效率,将其从35.3%提高到44.6%和从29.9%提高到37.3%.
- 确定crRNA序列长度是编辑效率的关键因素,20nt trucrRNAs被证明与24nt crRNAs一样有效,而较短的序列 (≤18nt) 的效率较低.
结论:
- CRISPR/LbCas12a系统是葡萄藤基因组工程的一个可行和有效的工具.
- 优化热处理和crRNA设计等条件,包括使用 trucrRNAs,提高了葡萄藤基因编辑的精度和效率.
- 这项研究为使用CRISPR/LbCas12a技术在葡萄藤中进行先进的基因操纵和作物改进铺平了道路.
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