Bircheneder Martin, Parniske Martin
Genetics, Faculty of Biology, LMU Munich, Grosshaderner Str. 2-4, D-82152, Martinsried, Germany.
Plant J. 2025 Sep;123(5):e70410. doi: 10.1111/tpj.70410.
While Cas9 and Cas12a are both RNA-guided endonucleases used for genome editing, only Cas12a is able to process pre-crRNA via its additional ribonuclease activity. This feature reduces the complexity of Cas12a versus Cas9-based genome editing systems thus providing an attractive alternative for generating site-specific mutations in plants. Here we aimed to improve the efficiency of the cas12a-based generation of two double-strand breaks flanking the open reading frame of a target gene, leading to its full deletion. To this end, we compared the relative impact of different components on cas12a-based gene deletion efficiency in three different eudicotyledons, Arabidopsis thaliana, Lotus japonicus, and Nicotiana benthamiana. We detected the highest cas12a-based editing efficiency with a combination of suitable promoters for crRNA and cas12a expression, a tandem terminator to control cas12a expression, a re-coded cas12a, adapted to the codon usage of Arabidopsis and engineered to carry introns, and encoding a Cas12a flanked by a nuclear localization signal at both ends. Our work revealed the high potential for improving cas12a-based genome editing systems for plant genetic research.
虽然Cas9和Cas12a都是用于基因组编辑的RNA引导的内切核酸酶，但只有Cas12a能够通过其额外的核糖核酸酶活性加工前体crRNA。这一特性降低了Cas12a与基于Cas9的基因组编辑系统相比的复杂性，从而为在植物中产生位点特异性突变提供了一个有吸引力的替代方案。在这里，我们旨在提高基于Cas12a在目标基因开放阅读框两侧产生两个双链断裂的效率，从而导致其完全缺失。为此，我们比较了不同组件对三种不同双子叶植物拟南芥、百脉根和本氏烟草中基于Cas12a的基因缺失效率的相对影响。我们检测到，当结合适合crRNA和Cas12a表达的启动子、用于控制Cas12a表达的串联终止子、重新编码的、适应拟南芥密码子使用情况并经过工程改造带有内含子且两端侧翼有核定位信号的Cas12a时，基于Cas12a的编辑效率最高。我们的工作揭示了改进基于Cas12a的基因组编辑系统用于植物遗传研究的巨大潜力。
