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在番茄细胞中比较Cas12a和Cas9介导的突变发生

Ellen Slaman^1,2, Lisanne Kottenhagen¹, William de Martines¹

¹Laboratory of Molecular Biology, Wageningen University & Research, Wageningen, The Netherlands.

Scientific reports

|February 24, 2024

概括

Lachnospiraceae (Lb) Cas12a是一种高效的CRISPR工具,用于番茄基因组编辑,表现出与SpCas9.9相比的强大性能. 这项研究描述了它的突变模式,并开发了用于常规应用的克隆系统.

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
基因组编辑基因组编辑

背景情况:

CRISPR-Cas12a具有独特的优势,如TTTV-PAM和特定的DNA裂变,使其成为基因组工程的宝贵工具.
描述Cas12a在番茄等植物中的有效性和特异性对于其在作物改良中的更广泛应用至关重要.

研究的目的:

评估Cas12a变种在番茄中进行基因组编辑的效率和特异性.
为 Cas12a crRNA 开发一个简单的克隆系统.
为了在番茄中比较Cas12a与SpCas9的目标和非目标活动.

主要方法:

番茄原塑体的高通量安普利康测序.
金门克隆用于crRNA构造.
对由LbCas12a和SpCas9.9诱导的目标和非目标突变的比较分析.

主要成果:

Lachnospiraceae (Lb) Cas12a在番茄基因组编辑方面表现出高效率.
与SpCas9.9相比,LbCas12a诱导了更多和更大的删除.
在57个部位中,在10个部位观察到LbCas12a的非目标突变,通常与PAM的远距离不匹配.

结论:

Cas12a是一种强大而多功能多功能的番茄基因组编辑工具,补充了SpCas9.
开发的黄金门系统有助于在番茄中进行常规的Cas12a应用.
在Cas12a中介的编辑通常是精确的,只要仔细考虑潜在的目标外站点.

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