Our project aims to develop an innovative in situ RNA sensing and regulation tool for yeast based on ADAR. To kickstart our idea, we constructed the tool in yeast and tested it in practical applications. Upon introducing the sensor and ADAR into the yeast, initial experimental results indicated low editing activity of ADAR, prompting a series of optimization efforts. Furthermore, two application scenarios - splice variant detection and starin security system - were chosen to assess the toolkit's capability in addressing real-world challenges.

我们的项目旨在开发一种基于ADAR的创新性酵母内RNA检测和调控工具。为了验证我们的想法，我们在酵母中构建了这个工具，并在实际应用中进行了测试。在将传感器和ADAR引入酵母后，我们预实验的结果表明ADAR的编辑活性较低，这促使我们进行了一系列的优化工作。此外，我们选择了两种应用场景——剪接异构体检测和工业菌株防盗门——来评估该工具在解决实际问题方面的能力。

Firstly, a ADAR system was constructed in yeast. In order to verify its activity, we designed four sensors which are control of each others. We added green fluorescence and blue fluorescence to both ends of the sensor. When termination codon existed, it could only express green fluorescent, when ADAR turned the termination codon into UIG, both green and blue fluorescence can be expressed.

首先，在酵母中构建了一个ADAR系统。为了验证其活性，我们设计了四个传感器，它们相互控制。我们在传感器的两端分别添加了绿色荧光和蓝色荧光。当存在终止密码子时，它只能表达绿色荧光；当ADAR将终止密码子转换成UIG时，绿色和蓝色荧光都可以表达。

However, the results of qPCR, western-blot and flow cytometry showed that the editing efficiency of ADAR in yeast was low. (Please refer to the Results section for detailed experimental results) So we then optimized this set of tools.

然而，qPCR、Western-blot和流式的结果显示，ADAR在酵母中的编辑效率很低（详见Results部分的详细实验结果）。因此，我们对我们的系统进行了优化。

First, we changed the promoter of ADAR from PDC to TEF, which is more potent, to increase the transcription of ADAR. In addition, after literature review, we found that addition of MS2 stem-loop sequence to ogRNA could improve the recruitment of RNA double-stranded regions to ADAR. We designed a set of control experiments to construct sensors with 0, 2, and 4 MS2 on ogRNA, respectively.

首先，我们将ADAR的启动子从PDC改为更为强大的TEF，以提高ADAR的转录水平。此外，在文献调研之后，我们发现在ogRNA上添加MS2井环结构序列可以增强RNA双链区域对ADAR的招募。我们设计了一组对照实验，分别构建了含有0、2和4个MS2的ogRNA的传感器。

qPCR results showed that the expression of TEF-ADAR was significantly higher than that of PDC-ADAR. The addition of the MS2 sequence also improved the editing efficiency of ADAR to some extent, as indicated by the flow cytometry results.

qPCR结果表明，TEF-ADAR的表达显著高于PDC-ADAR。MS2序列的添加也在一定程度上提高了ADAR的编辑效率，流式的结果也证明了这一点。

Splice variant plays a significant role in human diseases, particularly in the occurrence and progression of genetic disorders and cancers. Chk1 and Chk1s are variable splicing products of the CHK1 gene, and they play important roles in the regulation of the cell cycle and DNA damage checkpoint control. We choose Chk1 and Chk1s as our target gene, designing ogRNA at junctions where the two splice isoforms differed,testing whether ADAR could help us distinguish between two different splice isoforms.

剪接异构体在人类疾病中扮演着重要的角色，特别是在遗传性疾病和癌症的产生与演变中。Chk1和Chk1s是CHK1基因的不同剪接异构产物，它们在调节细胞周期和DNA损伤检查点控制中起着重要作用。我们选择Chk1和Chk1s作为我们的目标基因，设计了针对这两种剪接异构体不同的连接点的ogRNA，测试ADAR是否可以帮助我们区分两种不同的剪接异构体

We designed four control groups:

我们设计了4种control groups：

The brief experimental results are as follows.

以下是对我们的实验结果的简要展示。

In addition, we learned that enterprises have an urgent need for their own strain confidentiality and anti-theft, and the construction of a good burglar-proof door is conducive to protecting the patent of microbial strains. So we're trying to use ADAR to do the job of preventing theft. We placed a suicide gene BAX with loxp sites at both ends upstream of the Sensor and a Cre enzyme gene downstream. We selected GLC3 and the small heat shock protein HSP26 as target genes. Only when the culture temperature reaches the heat shock temperature, the Cre enzyme is expressed, the suicide gene is cleaved, and the bacteria can survive; otherwise, the suicide gene accumulates, and the cell initiates the suicide program.

此外，我们了解到企业对于自身菌株的保密性和防盗有迫切需求，构建良好的防盗门有利于保护微生物菌株的专利。因此，我们尝试使用ADAR来防止盗窃。我们在传感器上游两端放置了带有loxp位点的自杀基因BAX，下游放置了Cre酶基因。我们选择了GLC3和小热休克蛋白HSP26作为目标基因。只有当培养温度达到热休克温度时，Cre酶才会表达，自杀基因被切割，细菌才能存活；否则，自杀基因积累，细胞启动自杀程序。

We partially completed the plasmid construction, but subsequent experiments encountered resistance.

我们已经部分完成了质粒的构建，但随后的实验遇到了阻力。

1 Jiang, K., Liu, X., Wang, Y. et al. Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR. Nat Biotechnol 41, 698–707 (2023).
2 Sun, J., Shao, Z., Zhao, H. et al. Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae. Biotechnol Bioeng 160 (2012).
3 Pabla, N., Bhatt, K. & Dong, Z. Checkpoint kinase 1 (Chk1)-short is a splice variant and endogenous inhibitor of Chk1 that regulates cell cycle and DNA damage checkpoints. Proc Natl Acad Sci U S A 109, 197–202 (2012).
4 Wang, Z., Chatterjee, D., Jeon, H.Y. et al. Exon-centric regulation of pyruvate kinase M alternative splicing via mutually exclusive exons. J Mol Cell Biol 4, 79–87 (2012).

1 Jiang, K., Liu, X., Wang, Y. et al. Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR. Nat Biotechnol 41, 698–707 (2023).
2 Sun, J., Shao, Z., Zhao, H. et al. Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae. Biotechnol Bioeng 160 (2012).
3 Pabla, N., Bhatt, K. & Dong, Z. Checkpoint kinase 1 (Chk1)-short is a splice variant and endogenous inhibitor of Chk1 that regulates cell cycle and DNA damage checkpoints. Proc Natl Acad Sci U S A 109, 197–202 (2012).
4 Wang, Z., Chatterjee, D., Jeon, H.Y. et al. Exon-centric regulation of pyruvate kinase M alternative splicing via mutually exclusive exons. J Mol Cell Biol 4, 79–87 (2012).