Background

Expression of either Cas9 or Cas12a and guide RNAs by a single Polymerase II (Pol II) promoter represents a compact CRISPR expression system and has many advantages for different applications. In order to make this system routine in plant biology, engineering efforts are needed for developing and optimizing such single transcript unit (STU) systems for plant genome editing.

Results

To develop novel intron-based STU (iSTU) CRISPR system (STU CRISPR 3.0), we first evaluated three introns from three plant species for carrying guide RNAs by using an enhanced green fluorescence protein (eGFP) system in rice. After validation of proper intron slicing, we inserted these gRNA-containing introns into the open reading frames (ORFs) of Cas9 and Cas12a for testing their genome editing capability. Different guide RNA processing strategies have been tested for Cas9 and Cas12a. We demonstrated singular genome editing and multiplexed genome editing with these iSTU-Cas9 and iSTU-Cas12a systems.

Conclusion

We developed multiple iSTU-CRISPR/Cas9 and Cas12a systems for plant genome editing. Our results shed light on potential directions for further improvement of the iSTU systems.

中文翻译：

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基于内含子的单转录单位CRISPR系统，用于植物基因组编辑

背景

通过单个Polymerase II（Pol II）启动子表达Cas9或Cas12a和引导RNA代表了一个紧凑的CRISPR表达系统，并且对于不同的应用具有许多优势。为了使该系统成为植物生物学的常规方法，需要进行工程上的努力来开发和优化用于植物基因组编辑的此类单转录单位（STU）系统。

结果

为了开发基于内含子的新型STU（iSTU）CRISPR系统（STU CRISPR 3.0），我们首先通过在水稻中使用增强的绿色荧光蛋白（eGFP）系统评估了来自三种植物的三个内含子携带指导RNA。验证正确的内含子切片后，我们将这些含gRNA的内含子插入Cas9和Cas12a的开放阅读框（ORF），以测试其基因组编辑能力。已经针对Cas9和Cas12a测试了不同的指导RNA处理策略。我们通过这些iSTU-Cas9和iSTU-Cas12a系统演示了单基因组编辑和多基因组编辑。

结论

我们开发了多个iSTU-CRISPR / Cas9和Cas12a系统用于植物基因组编辑。我们的结果揭示了进一步改进iSTU系统的潜在方向。