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在Arabidopsis中提高编辑效率,其中LbCas12a变异携带D156R和E795L突变

Cuiping Xin¹, Dexin Qiao¹, Junya Wang¹

¹State Key Laboratory of Plant Environmental Resilience, College of Biological Sciences, China Agricultural University, Beijing, 100193 People's Republic of China.

|July 9, 2024

概括

研究人员设计了Cas12a变体,以改进Arabidopsis的基因组编辑. ttLbCas12a Ultra变种显示出高效率,为植物基因编辑提供了Cas9的有希望的替代品.

科学领域:

分子生物学分子生物学
植物生物技术植物生物技术
基因编辑技术的技术

背景情况:

CRISPR/Cas12a (Cpf1) 是一个类2型V核酶,具有编辑基因组的潜力.
目前的局限性包括温度敏感性和不充分的裂解活性,阻碍了广泛的应用.
Cas9是广泛使用的标准,但Cas12a提供了独特的优势.

研究的目的:

开发增强的Cas12a变体,以提高编辑效率和温度耐受性.
为了比较新型Cas12a变异与Arabidopsis中现有的变异的性能.
建立一个优越的Cas12a工具用于基因和促进器编辑在Arabidopsis.

主要方法:

通过结合耐温度和高活性变体的突变,产生了两种新的Cas12a变体,tAsCas12 Ultra和tTLbCas12a Ultra.
在四个Arabidopsis基因的六个目标位点中,比较了五种Cas12a变体 (LbCas12a, ttLbCas12a, ttLbCas12a Ultra, AsCas12a Ultra和 ttAsCas12 Ultra) 的编辑效率.
通过修改核定位信号序列和编码子使用,优化了表现最好的变体 (ttLbCas12a Ultra).

主要成果:

ttLbCas12a Ultra变种在22°C的温度下表现出了在Arabidopsis中所有测试变种中最高的编辑效率.
这种变异使得同卵性或双性突变物在一代内产生.
优化进一步显著提高了tTLbCas12a Ultra.的编辑效率.

结论:

ttLbCas12a Ultra是Cas9的高效和有价值的替代品,用于Arabidopsis的基因组编辑.
工程变体克服了温度灵敏度和裂变活性之前的限制.
该工具促进了植物中高效的基因和促进器编辑,加速了研究和育种.

关键词:

阿拉比多普西斯 (Arabidopsis) 是一种植物.这就是CRISPR/CasPR.这就是Cas12a.基因组编辑基因组编辑 ttLbCas12a 超级超级超级超级超级超级超级超级

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