In the pre-experiment, we constructed a total of 12 particles (an additional particle, pUC19-C1, was obtained directly from the laboratory) to observe the fluorescence using co-focus instruments, flow cellometers, and other instruments to calculate the editing effect of ADAR. Additionally, we have characterized ADAR using qPCR, western blot, and other methods to complete the basic conceptual validation.

在预实验中，我们构建了总共12个质粒（质粒pUC19-C1直接从实验室获得）并使用共聚焦仪器、流式细胞仪和其他仪器观察荧光，计算ADAR的编辑效果。此外，我们还使用qPCR、Western blot等方法对ADAR进行了表征，完成了基本概念验证。

When constructing a plasmid, we will first PCR amplify the gene fragments needed and then assemble them using the Gibson method. After the gibson assembly, we will introduce the constructed plasmid into E. coli and take a certain amount of bacterial solution to apply it to the Lysogeny broth to achieve the purpose of preserving, amplifying the plasmid, and screening the successfully transformed E. coli. The next morning, we will randomly take 8 single colonies from the medium applied the previous day for colony PCR and preservation of the strain. In the form of colony PCR, primers are designed near the interface during the gibson assembly, and PCR is performed to determine whether the gibson assembly is successful. The specific design of the primers can be seen in our attachment. After obtaining the results of colony PCR, we shake culture the bacteria whose bands are consistent with the expected results (positive results) so that they can be sent to the relevant company for sequencing on the third day to detect whether the target gene has produced possible non-synonymous mutations during the operation.

在构建质粒时，我们首先会PCR扩增所需的基因片段，然后使用Gibson方法组装它们。Gibson组装后，我们将构建的质粒导入大肠杆菌，并取一定量的菌液涂布到肉汤培养基上，以达到保存、扩增质粒和筛选成功转化的大肠杆菌的目的。第二天早上，我们会从前一天涂布的培养基上随机挑选8个单菌落进行菌落PCR和菌株保存。在菌落PCR中，我们设计了靠近Gibson组装界面的引物，并进行PCR以确定Gibson组装是否成功。具体的引物设计可以在我们的附件中看到。获得菌落PCR结果后，我们会摇培与预期结果一致的菌（阳性结果），以便在第三天将其送到相关公司进行测序，检测目标基因在操作过程中是否产生了可能的非同义突变。

In the following evidence section, we will explain all our constructed plasmids from four aspects: plasmid design, expected results, actual colony PCR results, and sequencing results.

在以下证据部分，我们将从四个方面解释我们构建的所有质粒：质粒设计、预期结果、实际菌落PCR结果和测序结果。

Step1. Design of plasmid

Step1. 质粒设计

This plasmid is mainly used to express ADAR1_p150 protein, that is, the optimized ADAR1 protein; AmpR and KanR are ampicillin resistance genes and kanamycin resistance genes, respectively, which are used for screening during transformation; FALG protein is mainly used as the target of the primary antibody in western blot during subsequent characterization.

该质粒主要用于表达ADAR1_p150蛋白，即优化的ADAR1蛋白；AmpR和KanR分别是氨苄青霉素抗性基因和卡那霉素抗性基因，用于转化过程中的筛选；FLAG蛋白主要作为后续表征过程中Western blot的一抗靶标。

Step2. Expected fragment length and expected electropherogram:

Step2. 预期片段长度和电泳结果：

port 1: 994bp primer：pCEVG-test-F1，ADAR1-test-R1
port 2: 1052bp primer：ADAR1-test-F2，pCEVG-test-R2

port 1: 994bp primer：pCEVG-test-F1，ADAR1-test-R1
port 2: 1052bp primer：ADAR1-test-F2，pCEVG-test-R2

Step3. Electrophoretic result:

Step3. 电泳结果：

We randomly picked 8 single colonies on the culture medium for colony PCR. (Remark: The marker we used in the pre-experiment had unclear bands for some reason, which we improved later.)

我们在培养基上随机挑选了8个单菌落进行菌落PCR。(备注：由于某些原因，我们在预实验中使用的标记物条带不清晰，后来我们对此进行了改进。)

Step4. Sequencing results:

Step4. 测序结果

The red arrows in Fig. 4 represent the sequencing results. The gaps or bulges on the arrows indicate deletions and mismatches, respectively. Since the length of the sequencing is approximately 1000 bp, but the results of the first and last 100 bp are inaccurate, we need to ensure that the first and last 100 bp of each sequencing segment are covered by the middle portions of other sequencing intervals (hence the overlap between our sequencing primers). This allows us to ignore inaccurate sequencing results and use accurate results when determining whether the sequencing sequence matches the designed sequence. The company's sequencing results (Fig. 4) show that our ADAR1_p150 gene contains only one synonymous mutation that does not affect the protein.

图4中的红色箭头代表测序结果。箭头上的间隙或凸起分别表示缺失和错配。由于测序长度约为1000bp，但首尾各100bp的结果不准确，我们需要确保每个测序片段的首尾100bp都被其他测序区间的中间部分所覆盖（因此我们的测序引物之间存在重叠）。这使我们能够忽略不准确的测序结果，并在确定测序序列是否与设计序列匹配时使用准确的结果。公司的测序结果（图4）显示，我们的ADAR1_p150基因只包含一个同义突变，不会影响蛋白质。

Step1. Design of plasmid

Step1. 质粒设计

This plasmid(Fig. 5) is mainly used to express ADAR2 protein connected to MCP. We also added NES nuclear export signal to this protein to facilitate ADAR2 to play an editing role in the cytoplasm.

这个质粒(图5)主要用于表达与MCP相连的ADAR2蛋白。我们还在这个蛋白上添加了NES核输出信号,以促进ADAR2在细胞质中发挥编辑作用。

Step2. Expected fragment length and expected electropherogram:

Step2. 预期片段长度和电泳结果：

port 1: 1058bp primer：pCEVG-test-F1，ADAR2-test-R1
port 2: 981bp primer：pCEVG-test-R2, ADAR2-test-F2

port 1: 1058bp primer：pCEVG-test-F1，ADAR2-test-R1
port 2: 981bp primer：pCEVG-test-R2, ADAR2-test-F2

Step3. Electrophoretic result:

Step3. 电泳结果：

The electrophoresis bands (Fig. 7) confirmed that the plasmids of bacteria No. 7 and No. 8 were successfully constructed.

电泳条带（图7）证实成功构建了7号和8号细菌的质粒。

Step4. Sequencing results:

Step4. 测序结果

The sequencing results shown below were correct.

根据下图，测序结果证明了我们构建成功。

This plasmid is a universal plasmid for yeast homologous recombination, and we obtained it directly from the laboratory.

该质粒是酵母同源重组的通用质粒，我们直接从实验室获得。

Step1. Design of plasmid

Step1. 质粒设计

This plasmid is mainly used to express the ADAR1_p150 protein and the optimized ADAR1 protein. Unlike pCEV-ADAR1, we aim to use pUC19-ADAR1-C2 to homologously recombine the gene expressing the ADAR1_150 protein into the yeast genome. However, due to the lengthy sequences of ADAR1_p150 and the marker gene, we utilize two fragments for homologous recombination. Therefore, in our design, HOR represents the part homologous to yeast, while the PDC1 promoter at the other end is homologous to pUC19-C1. The HOL of pUC19-C1 is another part homologous to yeast. We co-transform yeast with pUC19-C1 and pUC19-ADAR1-C2, allowing the marker gene and ADAR1_p150 to be integrated into the yeast genome together.

这个质粒主要用于表达ADAR1_p150蛋白和优化后的ADAR1蛋白。与pCEV-ADAR1不同，我们旨在使用pUC19-ADAR1-C2将表达ADAR1_150蛋白的基因通过同源重组整合到酵母基因组中。然而，由于ADAR1_p150和标记基因序列较长，我们采用两个片段进行同源重组。因此，在我们的设计中，HOR代表与酵母同源的部分，而另一端的PDC1启动子则与pUC19-C1同源。pUC19-C1的HOL是与酵母同源的另一部分。我们将pUC19-C1和pUC19-ADAR1-C2共转化到酵母中，使标记基因和ADAR1_p150能够一起整合到酵母基因组中。

Step2. Expected fragment length and expected electropherogram:

Step2. 预期片段长度和电泳结果：

port 1: 976bp primer：pUC19-test-F1，ADAR1-test-R1
port 2: 1020bp primer：ADAR2-test-F2， pUC19-test-R2

port 1: 976bp primer：pUC19-test-F1，ADAR1-test-R1
port 2: 1020bp primer：ADAR2-test-F2， pUC19-test-R2

Step3. Electrophoretic result:

Step3. 电泳结果：

The electrophoresis bands (Fig. 13) confirmed that the plasmid of strain No. 1 was successfully constructed.

电泳条带（图13）证实成功构建了菌株1的质粒。

Step4. Sequencing results:

Step4. 测序结果

The sequencing results shown below were correct.

根据下图，测序结果证明了我们构建成功。

Step1. Design of plasmid

Step1. 质粒设计

The plasmid design principle of pUC19-ADAR2_MCP-C2 is the same as that of pUC19-ADAR1-C2, both of which use homologous recombination to transfer marker genes and genes expressing ADAR proteins into the yeast genome.

pUC19-ADAR2_MCP-C2的质粒设计原理与pUC19-ADAR1-C2相同，两者都使用同源重组将标记基因和表达ADAR蛋白的基因转移到酵母基因组中。

Step2. Expected fragment length and expected electropherogram:

Step2. 预期片段长度和电泳结果：

port 1: 1400bp primer: PUC19-MCP-ADAR-test-F1, ADAR2-test-R1
port 2: 1007bp primer: ADAR2-test-F2, pUC19-test-R2

port 1: 1400bp primer: PUC19-MCP-ADAR-test-F1, ADAR2-test-R1
port 2: 1007bp primer: ADAR2-test-F2, pUC19-test-R2

Step3. Electrophoretic result:

Step3. 电泳结果：

The electrophoresis bands (Fig. 17) confirmed that the plasmids of strains 1 to 6 and 8 were successfully constructed.

电泳条带（图17）确认了1至6号和8号菌株的质粒已成功构建。

Step4. Sequencing results:

Step4. 测序结果

We selected No. 1 and No. 2 for sequencing. The sequencing results shown were correct.

我们选择了1号和2号进行测序。根据下图，测序结果证明了我们构建成功。

When we constructed pSensor1~4, we originally planned to use bleomycin for screening when transferring plasmids into yeast, but a yeast lawn appeared during our first transformation (too many yeasts on the culture medium could not pick a single colony for further culture). We repeated this experiment many times, trying strategies such as reducing the amount of bacterial solution applied, shortening the culture time, and increasing the concentration of bleomycin, but the yeast lawn still appeared. After further discussion, we speculated that it might be that the activity of bleomycin was weak or even ineffective, resulting in no screening effect on yeast, which in turn led to excessive yeast. Therefore, we used a culture medium lacking uracil in subsequent experiments and used the uracil synthesis gene as the marker gene of pSensor (our chassis organism, yeast strain B4742, does not have the uracil synthesis gene).

当我们构建pSensor1~4时，最初计划在将质粒转入酵母时使用博来霉素进行筛选，但在第一次转化时出现了酵母菌苔(培养基上的酵母太多，无法挑选单个菌落进行进一步培养)。我们多次重复了这个实验，尝试了减少涂布的菌液量、缩短培养时间、增加博来霉素浓度等策略，但酵母菌苔仍然出现。经过进一步讨论，我们推测可能是博来霉素的活性弱甚至失效，导致对酵母没有筛选作用，从而导致酵母过度生长。因此，在随后的实验中我们使用了缺乏尿嘧啶的培养基，并使用尿嘧啶合成基因作为pSensor的标记基因(我们的底盘生物，酵母菌株B4742，没有尿嘧啶合成基因)。

Step1. Design of plasmid

Step1. 质粒设计

The function of gRNA is to simulate the transcribed mRNA. According to our design, the transcribed gRNA can bind to the transcribed ogRNA to produce an A-C mismatch at the stop codon, and the UAG on the ogRNA will be edited by ADAR to UIG, so that transcription continues, and the blue fluorescent gene can be expressed. URA3 and AmpR are both marker genes used for screening.

gRNA的功能是模拟转录的mRNA。根据我们的设计，转录的gRNA可以与转录的ogRNA结合，在终止密码子处产生A-C错配，ogRNA上的UAG将被ADAR编辑为UIG，从而使转录继续进行，蓝色荧光基因得以表达。URA3和AmpR都是用于筛选的标记基因。

Since the failure of using bleomycin as a marker gene delayed our experimental progress, we decided to use the strategy of sending the remaining 4 sensor plasmids in the pre-experiment directly to the company for sequencing after the construction is successful to save time. Therefore, there is no expected fragment length, expected electrophoresis diagram and actual colony PCR band in the parts of these 4 plasmids.

由于使用博来霉素作为标记基因失败延误了我们的实验进度，我们决定采用在预实验中直接将剩余4个传感器质粒构建成功后送公司测序的策略以节省时间。因此，这4个质粒的部分没有预期片段长度、预期电泳图和实际菌落PCR条带。

Step2. Sequencing results:

Step2. 测序结果

Step1. Design of plasmid

Step1. 质粒设计

Compared with pSensor1_URA, we made a point mutation in the gRNA of pSensor2_URA, so that the gRNA will be fully complementary to the ogRNA without A-C mismatch. This design makes pSensor2_URA a negative control for pSensor1_URA, with the purpose of detecting the effect of mismatch on ADAR editing efficiency.

与pSensor1_URA相比，我们在pSensor2_URA的gRNA中进行了一个点突变，使gRNA与ogRNA完全互补，没有A-C错配。这个设计使pSensor2_URA成为pSensor1_URA的阴性对照，目的是检测错配对ADAR编辑效率的影响。

Step2. Sequencing results:

Step2. 测序结果

Step1. Design of plasmid

Step1. 质粒设计

pSensor3_URA does not contain gRNA that can complement ogRNA, but contains a plasmid called sensor. It is used as a negative control to detect the effect of dsRNA on ADAR editing efficiency.

pSensor3_URA不包含可以与ogRNA互补的gRNA，但包含一个称为sensor的质粒。它被用作阴性对照，用于检测双链RNA对ADAR编辑效率的影响。

Step2. Sequencing results:

Step2. 测序结果

Step1. Design of plasmid

Step1. 质粒设计

pSensor4_URA contains gRNA and edited ogRNA, that is, the termination codon UAG of the transcribed ogRNA has been changed to UIG. pSensor4_URA and 1 control can detect the editing efficiency of ADAR and serve as positive controls.

pSensor4_URA包含gRNA和已编辑的ogRNA，即转录的ogRNA的终止密码子UAG已被改变为UIG。pSensor4_URA和1对照可以检测ADAR的编辑效率，并作为阳性对照。

Step2. Sequencing results:

Step2. 测序结果

Since the design of our project is carried out in yeast, after successfully constructing the plasmid and storing it in E. coli, we need to extract a sufficient amount of plasmid from E. coli first, and then introduce the plasmid into the yeast by electroporation.

由于我们项目的设计是在酵母中进行的，在成功构建质粒并储存在大肠杆菌中后，我们需要先从大肠杆菌中提取足量的质粒，然后通过电穿孔将质粒导入酵母中。

During the culture process of yeast transformation, the homologous recombinant bacteria grew normally, but among the independent expression yeast, only the yeasts introduced with pCEV-ADAR1 and pSensor1_URA grew normally. We performed colony PCR on these microorganisms

在酵母转化的培养过程中，同源重组的细菌生长正常，但在独立表达的酵母中，只有导入了pCEV-ADAR1和pSensor1_URA的酵母生长正常。我们对这些微生物进行了菌落PCR.

To evaluate the success of the system we designed, we assessed the fluorescence intensity of each group. Initially, we utilized LSCM for detection.

为了评估我们设计的系统的成功与否，我们评估了每组的荧光强度。最初，我们使用LSCM进行检测。

Our sensor RNA construct consists of the eGFP sequence, the P2A peptide sequence, and the eBFP sequence. A termination codon (UAG) is located at the end of the eGFP sequence. When the sensor RNA is edited by ADAR, this termination codon is converted to UGG, which codes for tryptophan, allowing the translation to continue. The P2A peptide is utilized to facilitate the cleavage of the polypeptide chain, resulting in two separate fluorescent proteins that do not interfere with each other’s fluorescence. By measuring the fluorescence intensity and calculating the ratio of eBFP to eGFP, we can determine the editing efficiency of the ADARs.

我们的传感器RNA结构由eGFP序列、P2A肽序列和eBFP序列组成。在eGFP序列的末端有一个终止密码子(UAG)。当传感器RNA被ADAR编辑时，这个终止密码子被转换为UGG，编码色氨酸，允许翻译继续进行。P2A肽被用来促进多肽链的裂解，产生两个不会相互干扰荧光的独立荧光蛋白。通过测量荧光强度并计算eBFP与eGFP的比率，我们可以确定ADARs的编辑效率。

Additionally, we have established two negative control groups and one positive control group. In pSensor2, the gRNA has been mutated so that it can bind to the sensor RNA without an A-C mismatch. This control is set up to assess the impact of the A-C mismatch on ADAR editing. In pSensor3, the gRNA has been replaced with a different gRNA that does not form a double-stranded RNA (dsRNA) with our sensor RNA. This control group is designed to test the effect of dsRNA formation on ADAR editing. In pSensor4, the sensor RNA has been mutated such that the terminal codon UAG is already edited to UGG. This control group is used to confirm that the 2A peptide and the downstream eBFP are functioning correctly.

此外，我们还建立了两个阴性对照组和一个阳性对照组。在pSensor2中，gRNA已经突变，使其可以与传感器RNA结合而没有A-C错配。这个对照是为了评估A-C错配对ADAR编辑的影响。在pSensor3中，gRNA被替换为一个不与我们的传感器RNA形成双链RNA（dsRNA）的不同gRNA。这个对照组旨在测试dsRNA形成对ADAR编辑的影响。在pSensor4中，传感器RNA已经突变，使终止密码子UAG已经被编辑为UGG。这个对照组用于确认2A肽和下游的eBFP功能正常。

As shown in Fig. 33 and Fig. 44, the observation of blue fluorescence in both pSensor1 and pSensor4 suggests that both our ADAR and the sensor RNA are functional within yeast cells. However, the intensity of the blue fluorescence in pSensor1 is notably less than that in pSensor4. So we need to improve our sensing system to enhance its editing efficiency. To obtain more detailed results, we employed flow cytometry for further analysis.

如图33和图34所示，在pSensor1和pSensor4中都观察到蓝色荧光，表明我们的ADAR和传感器RNA在酵母细胞中都是功能正常的。然而，pSensor1中蓝色荧光的强度明显低于pSensor4。因此，我们需要改进我们的传感系统以提高其编辑效率。为了获得更详细的结果，我们使用流式细胞术进行了进一步分析。

Unfortunately, as shown in Fig. 36, for both ADAR1_p150 and ADAR2_MCP, between pSensor1 to pSensor3, we did not observe significant differences. While the eBFP/eGFP value of pSensor4 is close to 100%. The result showed that we need to improve the editing efficiency.

不幸的是，如图36所示，对于ADAR1_p150和ADAR2_MCP，从pSensor1到pSensor3,我们没有观察到显著差异。而pSensor4的eBFP/eGFP值接近100%。结果表明我们需要提高编辑效率。

To investigate the underlying causes of the low editing efficiency, we hypothesized that it might be attributed to the low expression level of the ADAR protein. Therefore, we conducted qPCR to measure the transcription level of the ADAR gene and Western Blot analysis to determine the expression level of the ADAR protein.

为了调查低编辑效率的潜在原因，我们假设这可能是由于ADAR蛋白的低表达水平造成的。因此，我们进行了qPCR来测量ADAR基因的转录水平，并进行了Western Blot分析来确定ADAR蛋白的表达水平。

Quantitative Polymerase Chain Reaction (qPCR), also known as real-time PCR, is a laboratory technique used to amplify and quantify DNA or RNA molecules. The technique utilizes fluorescent dyes or probes that bind to the DNA or RNA, allowing for real-time monitoring of the amplification process and quantification of the target sequence.

定量聚合酶链式反应（qPCR），也称为qPCR，是一种用于扩增和定量DNA或RNA分子的实验室技术。该技术利用与DNA或RNA结合的荧光染料或探针，允许实时监测扩增过程并定量目标序列。

For our qPCR experiment, we utilized ACT1 as the internal control gene. As shown in Figure 1, compared to ACT1, ADAR1's mRNA level is somewhat lower. ADAR2's mRNA level is comparable to ACT1's. Since ACT1 is expressed at a relatively low level in yeast cells, it is advisable to employ a stronger promoter in our subsequent modifications.

对于我们的qPCR实验，我们使用ACT1作为内部对照基因。如图1所示，与ACT1相比,ADAR1的mRNA水平略低。ADAR2的mRNA水平与ACT1相当。由于ACT1在酵母细胞中的表达水平相对较低，建议在后续修改中使用更强的启动子。

Western blot analysis was performed using polyacrylamide gel electrophoresis (PAGE), with the target molecules being proteins. The "probe" used was primary antibodies, while "detection" was achieved by employing secondary antibodies conjugated with markers. After separation by PAGE, the protein samples were transferred onto a solid-phase support, where proteins or peptides on the solid phase acted as antigens. These antigens were subjected to an immune reaction with corresponding primary antibodies, followed by a reaction with enzyme- or isotope-labeled secondary antibodies. The detection of electrophoretically separated specific protein components, which represent the expression of target genes, was then carried out via substrate color development or autoradiography.

采用聚丙烯酰胺凝胶电泳（PAGE）技术进行了蛋白质的Western blot分析。在此过程中，目标分子为蛋白质，所使用的“探针”为一抗，而”检测”则是通过使用与标记物结合的二抗来实现。蛋白质样品经PAGE分离后，转移至固相支持物上，其中固相上的蛋白质或肽段作为抗原。这些抗原随后与相应的一级抗体发生免疫反应，再与酶标记或同位素标记的二级抗体反应。通过底物显色反应或自动放射显影技术，检测电泳分离的特定蛋白质组分，这些组分代表了目标基因的表达。

We utilized Western blotting to characterize the expression levels of ADAR in yeast under different promoter strengths. The tags bound to primary antibodies for both ADAR1 and ADAR2 were FLAG. GADPH (30 kDa) was selected as the intracellular reference protein.

我们利用Western blotting技术对不同启动子强度下酵母中ADAR的表达水平进行了表征。ADAR1和ADAR2的一级抗体所结合的标签均为FLAG。选取了30 kDa的GADPH作为细胞内参照蛋白。

Due to changes in experimental progress and later experimental focus, we conducted two Western blot experiments

鉴于实验进程的变化以及后续实验焦点的调整，我们进行了两次Western blot实验

First experiment

第一次WB

In the first experiment, we inoculated and fermented three types of yeast expressing ADAR, along with a blank control, to allow the intracellular concentration of ADAR enzyme to reach a certain level. We then collected the cells by centrifugation and lysed them. Through subsequent centrifugation, each yeast sample was divided into three parts: lysate, supernatant, and pellet.

在首次实验中，我们接种并发酵了表达ADAR的三种酵母，并设置了一组空白对照，以使细胞内ADAR酶的浓度达到一定水平。随后，我们通过离心收集细胞并进行裂解。经过再次离心，每个酵母样品被分为三部分：裂解液、上清液和沉淀物。

Second experiment

第二次实验

During the experiment, we observed that the growth of yeast strains expressing ADAR independently was severely restricted. We hypothesize that ADAR may have some toxicity, and that the highest expression levels were achieved using plasmid-based ADAR expression. Therefore, in subsequent experiments, we abandoned the independent expression format. Based on the experience from the previous experiment, we also found that the ADAR enzyme was primarily present in the supernatant of the yeast lysates. Therefore, in the second experiment, we only fractionated one yeast strain into two samples: lysate and supernatant.

在实验过程中，我们观察到独立表达ADAR的酵母菌株生长受到严重限制。我们推测ADAR可能具有一定的毒性，并且基于质粒的ADAR表达实现了最高表达水平。因此，在后续实验中，我们放弃了独立表达的形式。根据前一次实验的经验，我们还发现ADAR酶主要存在于酵母裂解液的上清液中。因此，在第二次实验中，我们仅将一个酵母菌株分为两个样本：裂解液和上清液。

Conclusion and Discussion

结论与讨论

In both experiments, the ADAR1_p150 band was clearly visible, but we did not observe a band for ADAR2_MCP. However, in other experiments, we found that systems involving ADAR2_MCP exhibited high editing efficiency (compared to the blank control), which confirms that ADAR2_MCP was indeed expressed in the yeast. It was simply not detected in the Western blot results. To explain this, we propose the following hypotheses, We will design follow-up experiments to further test these hypotheses.

1. ADAR2_MCP may have high intracellular activity but is rapidly degraded outside the cell, and the prolonged duration of the Western blot procedure may not favor the long-term stability of ADAR2_MCP.

2. Another possibility is that the spatial structure of ADAR2_MCP is such that the FLAG tag at the protein's terminus may be folded into the interior of the protein, making it difficult for the primary antibody to bind.

在两次实验中，ADAR1_p150的条带清晰可见，但我们没有观察到ADAR2_MCP的条带。然而，在其他实验中，我们发现涉及ADAR2_MCP的系统展现出较高的编辑效率（与空白对照相比），这证实了ADAR2_MCP确实在酵母中得到了表达。只是在Western blot结果中未检测到。为了解释这一现象，我们提出了以下假设，并将设计后续实验以进一步验证这些假设。

1. ADAR2_MCP可能在细胞内具有较高的活性，但在细胞外迅速降解，而Western blot程序的延长可能不利于ADAR2_MCP的长期稳定性。

2. 另一种可能性是ADAR2_MCP的空间结构使得蛋白质末端的FLAG标签可能折叠到蛋白质内部，从而使得一级抗体难以结合。

We hypothesize that the subcellular localization of ADAR proteins may influence their RNA editing efficiency. Given that ogRNA is localized in the cytoplasm, ADAR proteins confined to the nucleus may have limited access to cytoplasmic ogRNA, thereby reducing editing efficiency. To investigate this, we employed a linker (GGGGS linker) sequence to fuse ADAR proteins with the red fluorescent protein mScarlet, enabling the visualization of ADAR1 and ADAR_MCP localization within the cell. We subsequently constructed two plasmids and integrated the target genes into yeast cells through homologous recombination, followed by fluorescence observation using laser scanning confocal microscopy (LSCM). Our results demonstrated that ADAR1 predominantly localized to the nucleus, with minor cytoplasmic distribution, while ADAR_MCP was primarily cytoplasmic.

我们假设ADAR蛋白的亚细胞定位可能影响其RNA编辑效率。鉴于ogRNA定位在细胞质中，限制在细胞核中的ADAR蛋白可能难以接触到细胞质中的ogRNA，从而降低编辑效率。为了研究这一点，我们使用连接子(E2A肽)序列将ADAR蛋白与红色荧光蛋白mScarlet融合，使ADAR1和ADAR_MCP的定位在细胞内可视化。随后，我们构建了两个质粒，通过同源重组将目标基因整合到酵母细胞中，然后使用激光扫描共焦显微镜(LSCM)进行荧光观察。我们的结果表明，ADAR1主要定位于细胞核，少量分布在细胞质中，而ADAR_MCP主要分布在细胞质中。

To determine the localization of ADAR1 and ADAR_MCP, we constructed two plasmids, pLocate-ADAR1 and pLocate-ADAR_MCP. Each plasmid utilized a linker to fuse ADAR with mScarlet. We can determine the location of the fluorescent protein by LSCM, and then determine the location of the ADAR protein. Illustrations of the plasmid designs are provided below.

为了确定ADAR1和ADAR_MCP的定位，我们构建了两个质粒，pLocate-ADAR1和pLocate-ADAR_MCP。每个质粒都使用连接子将ADAR与mScarlet融合。我们可以通过LSCM确定荧光蛋白的位置，然后确定ADAR蛋白的位置。下面提供了质粒设计的示意图。

Plasmid construction

质粒构建

Following Gibson assembly of the plasmids, we transformed *E. coli*. The next day, we selected eight single colonies from LB medium for colony PCR using the following primers:

在质粒的Gibson组装之后，我们转化了大肠杆菌。第二天，我们从LB培养基中选择了八个单菌落进行菌落PCR，使用以下引物：

pLocate-ADAR1: ADAR1-Scarlet-idenF and ADAR2-Scarlet-idenR
pLocate-ADAR_MCP: ADAR2-Scarlet-idenF and ADAR2-Scarlet-idenR

pLocate-ADAR1: ADAR1-Scarlet-idenF and ADAR2-Scarlet-idenR
pLocate-ADAR_MCP: ADAR2-Scarlet-idenF and ADAR2-Scarlet-idenR

The expected length of both is about 1000bp.

预期的长度都约为1000bp。

Electrophioult:

电泳

In the electrophoresis diagram of ADAR1, we can see that in addition to the band at about 1000bp, there is another band at 250bp. This may be because when we obtained the plasmid backbone by PCR and recovered the DNA, the length of the backbone was close to the length of the entire plasmid, which led to the introduction of part of the original plasmid. Due to the primer design of our colony PCR (which can be viewed in the map), the original plasmid without mScarlet will also produce a band during the colony PCR process, and the length is exactly 1000-693=307, near the 250bp band.

在ADAR1的电泳图中，我们可以看到除了约1000bp的条带外，还有一个250bp的条带。这可能是因为我们通过PCR获得质粒骨架并回收DNA时，骨架的长度接近整个质粒的长度，导致引入了部分原始质粒。由于我们菌落PCR的引物设计(可以在图中查看)，没有mScarlet的原始质粒在菌落PCR过程中也会产生一个条带，长度恰好为1000-693=307，接近250bp条带。

For pLocate-ADAR_MCP, the observed band was around 750 bp instead of the expected 1000 bp, which may have been due to incorrect primer addition or slight electrophoretic shifts. Nevertheless, subsequent sequencing confirmed the correct assembly of the plasmids.

对于pLocate-ADAR_MCP，观察到的条带约为750 bp而不是预期的1000 bp，这可能是由于引物添加不正确或电泳轻微偏移造成的。不过，随后的测序确认了质粒是正确组装的。

The sequencing results show that the plasmid construction is correct.

测序结果表明质粒构建正确。

Yeast Transformation

酵母转化

Following plasmid construction, we proceeded with yeast transformation to integrate the target genes into the yeast genome via homologous recombination. We utilized the pUC19-C1 plasmid for this purpose.

质粒构建完成后，我们进行了酵母转化，通过同源重组将目标基因整合到酵母基因组中。我们为此使用了pUC19-C1质粒。

As shown in the Fig.46, we first linearize the plasmid by PCR to obtain C1 and C2 fragments (same principle as in Pre-experiment, C1 is fixed, while C2 contains our target gene), and then transfer C1 and C2 fragments into yeast by electroporation for homologous recombination. DNA fragments containing homologous segments can be combined with each other, and finally the target gene is homologously recombined into the yeast genome. After electroporation, we inoculated the bacterial solution into SD-His medium for screening. After two days, we performed colony PCR on the yeast colonies. Given the limited number of colonies, we screened as many as possible.

如图46所示，我们首先通过PCR线性化质粒以获得C1和C2片段（原理与预实验相同，C1是固定的，而C2包含我们的目标基因），然后通过电转将C1和C2片段转移到酵母中进行同源重组。含有同源片段的DNA片段可以相互结合，最终目标基因通过同源重组整合到酵母基因组中。电转后，我们将菌液接种到SD-His培养基中进行筛选。两天后，我们对酵母菌落进行了菌落PCR。由于菌落数量有限，我们尽可能多地进行了筛选。

Out of the selected colonies, four of the pLocate-ADAR1 transformants (including ADAR1-ms) and one of the pLocate-ADAR_MCP transformants (including ADAR2-ms) were confirmed to be correct. These correct clones were cultured overnight, followed by a short incubation to optimize yeast growth for fluorescence observation.

在选择的菌落中，四个pLocate-ADAR1转化子(包括ADAR1-ms)和一个pLocate-ADAR_MCP转化子(包括ADAR2-ms)被确认为正确的。这些正确的克隆过夜培养后，进行了短时间培养以优化酵母生长，为荧光观察做准备。

For ADAR1, several cells exhibited one or more "red bright spots" (e.g., Figure 49, B2), likely corresponding to the yeast nucleus, with some fluorescence dispersed throughout the cytoplasm. This suggests that ADAR1 is predominantly nuclear, with some cytoplasmic distribution, potentially accounting for the reduced editing efficiency observed in preliminary experiments.

对于ADAR1，几个细胞显示出一个或多个"红色亮点"(例如，图49，B2)，可能对应于酵母细胞核，同时一些荧光分散在整个细胞质中。这表明ADAR1主要位于细胞核，但也有一些分布在细胞质中，这可能解释了初步实验中观察到的较低编辑效率。

Conversely, ADAR_MCP exhibited a clear exclusion of fluorescence from the nuclear region, indicating predominant cytoplasmic localization. This may be attributed to the presence of a nuclear export signal (NES) added to ADAR_MCP.

相反，ADAR_MCP在核区域明显排除了荧光，表明主要定位于细胞质。这可能归因于添加到ADAR_MCP中的核输出信号(NES)。

Fluorescence microscopy results are presented below:

荧光显微镜结果如下所示：

During the cell culture process, we observed varying degrees of turbidity in different yeast strains under the same culture duration. This led us to suspect that ADAR may have cytotoxic effects, potentially inhibiting yeast growth. Thus, we designed follow-up experiments to verify this hypothesis.

在细胞培养过程中，我们观察到在相同培养时间下，不同酵母菌株的浑浊度有所不同。这让我们怀疑ADAR可能具有细胞毒性，潜在地抑制酵母生长。因此，我们设计了后续实验来验证这一假设。

We plan to compare the effects of different ADAR proteins on yeast growth, as well as the effects of the same ADAR at different expression levels. We selected four yeast strains that have ADAR genes homologously recombined into their genomes, along with a blank control group. Using different promoters, we control ADAR expression intensity. We will dilute the activated cultures to the same concentration and spot them onto solid culture plates for the same amount of time, allowing us to observe colony growth rates and characterize the toxicity of ADAR to yeast cells.

我们计划比较不同ADAR蛋白对酵母生长的影响，以及同一ADAR在不同表达水平下的影响。我们选择了四种已将ADAR基因同源重组到基因组中的酵母菌株，以及一个空白对照组。通过使用不同的启动子，我们控制ADAR的表达强度。我们将激活的培养物稀释到相同浓度，并将它们点种到固体培养板上相同时间，这样我们就可以观察菌落生长速率并表征ADAR对酵母细胞的毒性。

The growth status of the five groups was recorded after 13h, 19h, 24h, and 36h as shown in the following images:

记录了五组在13h、19h、24h和36h后的生长状况，如下图所示:

Due to the rapid growth of group B and the slow growth of group T, it was challenging to directly compare the growth rates across all five groups. Therefore, we focused on the second and third groups. It was observed that the four yeast strains with ADAR grew more slowly than the blank control, suggesting that ADAR1_p150 and ADAR2_MCP have inhibitory effects on cell growth.

由于B组生长迅速而T组生长缓慢，难以直接比较五组的生长速率。因此，我们主要关注第二和第三组。观察到，四个含ADAR的酵母菌株比空白对照生长更慢，表明ADAR1_p150和ADAR2_MCP对细胞生长有抑制作用。

Interestingly, yeast strains with stronger promoters showed faster growth compared to those with weaker promoters for the same ADAR, which is counterintuitive. Typically, the toxicity of a substance would correlate positively with its concentration, making these results difficult to explain with current data.

有趣的是，对于相同的ADAR，强启动子的酵母菌株比弱启动子的生长更快，这与直觉相反。通常，物质的毒性与其浓度呈正相关，使得这些结果难以用现有数据解释。

This experiment was a repeat of a previous preliminary experiment. Compared to the pre-experiment, only the colony composition was optimized, with all other parameters and procedures unchanged. Figure 55 shows the colony morphology in the preliminary experiment at OD600=0.001 after 26 hours of incubation. The results showed that:

1. the yeast strains with ADAR grew more slowly than the control

2. ADAR1_p150 strains with a strong promoter grew faster than those with a weak promoter.

These findings are consistent with our current experiment.

本实验是对之前初步实验的重复。与预实验相比，仅优化了菌落组成，其他参数和程序保持不变。图55显示了预实验中OD600=0.001的菌落在26小时培养后的形态。结果显示：

1. 含ADAR的酵母菌株生长比对照组慢

2. 强启动子的ADAR1_p150菌株生长比弱启动子的快

这些发现与我们当前的实验一致。

The primary difference between the two experiments was that in the preliminary experiment, the ADAR2_MCP strain with the TEF promoter exhibited a severe inhibitory effect on yeast growth, whereas in the current experiment, the TEF promoter promoted faster growth compared to the weak promoter. Referring to other team members' results, T2 yeast does indeed grow slower under similar conditions, aligning more closely with the pre-experiment results. This suggests that there may have been an error in the preparation of the T2 strain during this toxicity experiment, potentially leading to an over-concentration of the T2 solution.

Both experiments also showed a negative correlation between ADAR1_p150 toxicity and expression intensity, which remains unexplained. We propose two possible explanations:

两次实验的主要区别在于，在预实验中，TEF启动子的ADAR2_MCP菌株对酵母生长表现出严重的抑制作用，而在本次实验中，TEF启动子促进了比弱启动子更快的生长。参考其他团队成员的结果，T2酵母在类似条件下确实生长较慢，更接近预实验结果。这表明在本次毒性实验中T2菌株的制备可能存在错误，可能导致T2溶液过度浓缩。

两次实验还都显示ADAR1_p150毒性与表达强度呈负相关，这仍然无法解释。我们提出两种可能的解释：

1. The results are accurate, and the relationship between ADAR1_p150 expression intensity and toxicity is complex, requiring further exploration of its metabolic impact in yeast cells.

1. 结果准确，ADAR1_p150表达强度与毒性的关系复杂，需要进一步探究其在酵母细胞中的代谢影响。

2. After consulting our advisor, it was suggested that long-term storage at -80°C reduces glycerol stock activity, necessitating a longer activation time. To mitigate this, we plan to plate the glycerol stocks on solid medium for activation and select single colonies to repeat the experiment, thereby minimizing interference from refrigeration.

2. 咨询我们的指导老师后，建议-80°C长期储存会降低甘油菌株活性，需要更长的激活时间。为缓解这一问题，我们计划将甘油菌株涂布在固体培养基上激活，并选择单个菌落重复实验，从而最大限度地减少冷藏带来的干扰。

Conclusion: Both ADAR1_p150 and ADAR_MCP inhibit yeast growth, but our current experiments cannot fully explain the relationship between ADAR expression intensity and its toxicity. It is necessary to follow our advisor's advice, eliminate strain activity interference from refrigeration, and conduct new experiments to clarify this relationship.

结论：ADAR1_p150和ADAR_MCP都抑制酵母生长，但我们目前的实验无法完全解释ADAR表达强度与其毒性的关系。有必要按照指导老师的建议，消除冷藏对菌株活性的干扰，并进行新的实验以阐明这种关系。

In summary, the preliminary experiment basically proved that our RNAssay is feasible. Both ADAR1 and ADAR_MCP are correctly expressed in yeast and play certain functions, but the editing efficiency is relatively low. Subcellular localization experiments show that ADAR1 is mostly located in the nucleus, while ADAR_MCP is mainly located in the cytoplasm. The toxicity test of ADAR proves that ADAR does have certain cytotoxicity. In the future, we will further optimize the editing efficiency of ADAR based on the preliminary experiment and develop some useful applications. Therefore, we divided into 3 groups in the subsequent experiments, one group for ADAR optimization, and the other 2 groups for application development.

总之，初步实验基本证明了我们的RNAssay是可行的。ADAR1和ADAR_MCP都在酵母中正确表达并发挥一定功能，但编辑效率相对较低。亚细胞定位实验表明ADAR1主要位于细胞核，而ADAR_MCP主要位于细胞质。ADAR的毒性测试证明ADAR确实具有一定的细胞毒性。未来，我们将基于初步实验进一步优化ADAR的编辑效率，并开发一些有用的应用。因此，在后续实验中我们分为3组，一组用于ADAR优化，另外2组用于应用开发。

After obtaining preliminary experimental results, we realized that the editing efficiency of ADAR was relatively low. Therefore, we attempted the following two improvements:

1. Replacing the promoter with the stronger TEF promoter

2. increasing the number of MS2 sites around the binding region, with quantities of 0, 2, and 4 respectively

After optimizing the ADAR editing efficiency, we designed a plasmid induced by xylose expression. By measuring the editing efficiency and the expression level of the target transcript, we can semi-quantitatively detect the expression level of the target transcript.

在获得初步实验结果后，我们意识到ADAR的编辑效率相对较低。因此，我们尝试了以下两项改进：。

1. 用更强的TEF启动子替换原有启动子

2. 增加结合区域周围的MS2位点数量，分别为0、2和4个

在优化ADAR编辑效率后，我们设计了一个由木糖诱导表达的质粒。通过测量编辑效率和目标转录本的表达水平，我们可以半定量地检测目标转录本的表达水平。

We constructed 3 plasmids that replaced the PDC promoter with the TEF promoter: TEF-C1, TEF-C2-ADAR1,TEF-C2-ADAR_MCP.

我们构建了3个将PDC启动子替换为TEF启动子的质粒：TEF-C1、TEF-C2-ADAR1、TEF-C2-ADAR_MCP。

plasmid construction

质粒构建

Step1. Design of plasmid :

质粒设计：

The plasmid is basically the same as pUC19-C1, pUC19-ADAR1-C2, and pUC19-ADAR_MCP-C2. We only replaced the PDC1 promoter with the TEF1 promoter, and the homologous regions of C1 and C2 were also changed.

质粒与pUC19-C1、pUC19-ADAR1-C2和pUC19-ADAR_MCP-C2基本相同。我们只将PDC1启动子替换为TEF1启动子，C1和C2的同源区域也相应改变。

Step2. Sequencing results:

测序结果：

The company's sequencing results show that our construction of TEF-C1, TEF-C2-ADAR1 and TEF-C2-ADAR_MCP plasmid is completely right.

公司的测序结果显示我们构建的TEF-C1、TEF-C2-ADAR1和TEF-C2-ADAR_MCP质粒完全正确。

To detect whether the transcription level of ADAR gene was changed after the replacement of promoter, We extracted total RNA from yeast transformed with either PDC1-ADAR and TEF1-ADAR, then measured it using qPCR.

为检测启动子替换后ADAR基因的转录水平是否发生变化，我们从转化了PDC1-ADAR和TEF1-ADAR的酵母中提取总RNA，然后使用qPCR进行测量。

The validation results demonstrate that both ADAR1 and ADAR2 were successfully introduced into yeast. Additionally, the use of the TEF1 promoter resulted in significantly elevated ADAR gene transcription levels compared to the original PDC-1 promoter, with the differences being statistically significant.

验证结果表明，ADAR1和ADAR2都成功引入酵母。此外，使用TEF1启动子导致ADAR基因转录水平显著高于原始PDC-1启动子，差异具有统计学意义。

Literature suggests that adding MS2 sequences on both sides of the binding region can improve ADAR editing efficiency. Therefore, we designed three types of sensors with 0, 2, and 4 MS2 sequences around the binding region. In our experimental design, we introduced two parts into yeast. The first part is the ADAR protein expression, with four gene types: PDC promoter + ADAR1, PDC promoter + ADAR_MCP, TEF promoter + ADAR1, and TEF promoter + ADAR_MCP. The second part is the sensor, with three types containing 0, 2, or 4 MS2 sequences. The overall design is shown in the diagram. This method allows us to determine the combination with the highest editing efficiency. After construction, we observed these 12 engineered yeast strains using confocal microscopy and flow cytometry to calculate ADAR editing efficiency.

文献表明，在结合区域两侧添加MS2序列可以提高ADAR编辑效率。因此，我们设计了三种传感器，在结合区域周围分别含有0、2和4个MS2序列。在我们的实验设计中，我们将两个部分引入酵母。第一部分是ADAR蛋白表达，有四种基因类型：PDC启动子 + ADAR1、PDC启动子 + ADAR_MCP、TEF启动子 + ADAR1和TEF启动子 + ADAR_MCP。第二部分是传感器，有三种类型，分别含有0、2或4个MS2序列。总体设计如图所示。这种方法使我们能够确定具有最高编辑效率的组合。构建完成后，我们使用共焦显微镜和流式细胞仪观察这12种工程酵母菌株，以计算ADAR编辑效率。

Plasmid with added MS2(0/2/4): pSensor-MS2-0, pSensor-MS2-2, pSensor-MS2-4

添加MS2(0/2/4)的质粒：pSensor-MS2-0、pSensor-MS2-2、pSensor-MS2-4

plasmid construction

质粒构建

Step1. Design of plasmid :

质粒设计：

As mentioned earlier, we added 0, 2, and 4 MS2 sequences around the RNA complementary pairing structure.

如前所述，我们在RNA互补配对结构周围添加了0、2和4个MS2序列。

Step2. Sequencing results:

测序结果：

The company's sequencing results show that our construction of pSensor-MS2-0, pSensor-MS2-2, pSensor-MS2-4 plasmid is completely right.

公司的测序结果显示我们构建的pSensor-MS2-0、pSensor-MS2-2、pSensor-MS2-4质粒完全正确。

Successful yeast transformation

酵母转化成功

Yeast transformation was successful

酵母成功完成转化

We performed confocal fluorescence microscopy imaging and sorted the cells using flow cytometry in order to calculate the editing efficiency.

我们进行了共焦荧光显微镜成像，并使用流式细胞仪对细胞进行分选，以计算编辑效率。

Figures 64 and 65 clearly show that the addition of MS2 sequences effectively enhances the editing efficiency of the entire system. However, an unexpected observation is that increasing the number of MS2 sequences leads to a decrease in editing efficiency for both TEF-ADAR1 and TEF-ADAR2. This outcome contrasts with the results observed for PDC-ADAR2. We hypothesize that the MS2 sequences may interfere with the transcription process, potentially causing premature termination of the mScarlet-EGFP fusion protein. When ADAR1/2 is driven by the PDC promoter, the number of MS2 sequences correlates with increased binding affinity, leading to enhanced editing efficiency. In contrast, under the control of the TEF promoter, the high expression levels of ADAR1/2 may lead to saturation, making the concentration of sensor RNA a limiting factor. Given that MS2 sequences might increase the likelihood of abortive transcription, a higher number of MS2 sequences could reduce the available concentration of sensor RNA, thereby decreasing editing efficiency.

图64和65清楚地表明，添加MS2序列有效地提高了整个系统的编辑效率。然而，一个意外的观察结果是，增加MS2序列的数量导致TEF-ADAR1和TEF-ADAR2的编辑效率降低。这一结果与PDC-ADAR2观察到的结果相反。我们假设MS2序列可能会干扰转录过程，可能导致mScarlet-EGFP融合蛋白的提前终止。当ADAR1/2由PDC启动子驱动时，MS2序列的数量与结合亲和力增加相关，导致编辑效率提高。相比之下，在TEF启动子的控制下，ADAR1/2的高表达水平可能导致饱和，使传感器RNA的浓度成为限制因素。考虑到MS2序列可能增加中止转录的可能性，更多的MS2序列可能会降低可用的传感器RNA浓度，从而降低编辑效率。

Furthermore, Figures 66 and 67 highlight that TEF-ADAR2 exhibits the highest editing efficiency, likely attributable to its elevated expression level.

此外，图66和67突出显示TEF-ADAR2表现出最高的编辑效率，这可能归因于其较高的表达水平。

plasmid construction

质粒构建

Step1. Design of plasmid :

质粒设计：

We designed a xylose-inducible promoter upstream of the target transcript. Under low xylose concentrations, xylR can inhibit the transcription of the xylose promoter. However, when the xylose concentration increases, xylose binds to xylR, causing it to lose its repressive activity and thus promoting the expression of dow nstream genes. The other parts of the plasmid remain essentially the same as before, so they are not elaborated here.

我们在目标转录本的上游设计了一个木糖诱导启动子。在低木糖浓度下，xylR可以抑制木糖启动子的转录。然而，当木糖浓度增加时，木糖与xylR结合，导致其失去抑制活性，从而促进下游基因的表达。质粒的其他部分基本与之前相同，所以这里不再赘述。

We will integrate the target gene into the yeast genome through homologous recombination.

我们将通过同源重组将目标基因整合到酵母基因组中。

Step2. Sequencing results:

测序结果：

Based on our initial results, no clear relationship was observed between the target transcript concentration and editing efficiency. We hypothesized that this could be attributed to an insufficient induction period. Consequently, we planned to extend the induction time from 12 hours to 24 hours in subsequent experiments.

根据我们的初步结果，目标转录本浓度与编辑效率之间没有观察到明显的关系。我们假设这可能是由于诱导时间不足造成的。因此，我们计划在后续实验中将诱导时间从12小时延长到24小时。

However, in our next experiment, we were unable to detect red fluorescence under the microscope, suggesting a potential issue with homologous recombination, despite obtaining correct results in the colony PCR. Due to the extended duration of the experiment and the time constraints imposed by the start of the new semester, we were unable to repeat the experiment at this stage. Moving forward, we aim to obtain a more robust dataset in order to enhance the overall completeness of our project.

然而，在我们的下一个实验中，我们无法在显微镜下检测到红色荧光，这表明同源重组可能存在问题，尽管我们在菌落PCR中获得了正确的结果。由于实验时间延长以及新学期开始带来的时间限制，我们无法在这个阶段重复实验。展望未来，我们的目标是获得更加稳健的数据集，以提高我们项目的整体完整性。

Our design primarily employs an unexpected unlocking mechanism to protect yeast strains from unauthorized acquisition. Typically, yeast is cultured at 30°C with sufficient nutrients, which often include glucose. However, in our system, if the yeast is not cultured at 40°C or glucose is present in the medium, the suicide gene will be activated, leading to the death of the yeast.

我们的设计主要采用一种意想不到的解锁机制来保护酵母菌株不被未经授权获取。通常，酵母在30°C下培养，有足够的营养，其中通常包括葡萄糖。然而，在我们的系统中，如果酵母不在40°C下培养或培养基中存在葡萄糖，自杀基因将被激活，导致酵母死亡。

The underlying principle is as follows: HSP26 is an endogenous yeast gene whose expression at 40°C is 160 times higher than at 30°C, while GLC3 is highly expressed in the absence of external glucose. GSDMD and BAX are the selected suicide genes in our system, promoting pyroptosis and apoptosis, respectively. These suicide genes are constitutively expressed. The targetRNA is designed with an ogRNA on the sensor that can form a dsRNA region with either HSP26 or GLC3. The suicide genes are flanked by two loxP sequences, resulting in four distinct "security locks" due to the variation in ogRNA and the corresponding suicide genes.

基本原理如下：HSP26是一种内源性酵母基因，其在40°C下的表达量是30°C下的160倍，而GLC3在外部没有葡萄糖时高度表达。GSDMD和BAX是我们系统中选择的自杀基因，分别促进细胞焦亡和细胞凋亡。这些自杀基因被持续表达。靶标RNA被设计在传感器上有一个ogRNA，可以与HSP26或GLC3形成dsRNA区域。自杀基因被两个loxP序列包围，由于ogRNA和相应自杀基因的变化，产生了四种不同的"安全锁"。

When the yeast is cultured at 40°C (similarly when glucose is present in the medium), the high transcription level of HSP26 leads to the formation of an A-C mismatch dsRNA with the ogRNA region, which recruits ADAR to edit the stop codon UAG to UIG. This enables the downstream Cre recombinase gene to be expressed, excising the suicide genes located between the two loxP sites and "unlocking" the protective mechanism. However, if the yeast is cultured solely at 30°C, the suicide genes will not be excised, and since they are constitutively expressed, the yeast will quickly perish, preventing the unauthorized expansion of the strain.

当酵母在40°C下培养时（类似地，当培养基中存在葡萄糖时），HSP26的高转录水平导致与ogRNA区域形成A-C错配的dsRNA，这招募ADAR将终止密码子UAG编辑为UIG。这使得下游的Cre重组酶基因得以表达，切除位于两个loxP位点之间的自杀基因，从而"解锁"保护机制。然而，如果酵母仅在30°C下培养，自杀基因将不会被切除，由于它们被持续表达，酵母将很快死亡，防止未经授权的菌株扩增。

We plan to integrate the target gene into the yeast genome by homologous recombination. At present, we have achieved the construction of pSensor-URA-Cre plasmid and other plasmid fragments.

我们计划通过同源重组将目标基因整合到酵母基因组中。目前为止，我们实现了pSensor-URA-Cre质粒以及其他质粒片段的构建。

The sequencing results of pSensor-URA-Cre plasmid are as follows.

pSensor-URA-Cre质粒的测序结果如下。

However, since we needed to construct the plasmid through six-fragment Gibson assembly, it was difficult and had a low success rate. At the same time, the laboratory needed to be renovated, which resulted in insufficient experimental time, so we were ultimately unable to complete the construction of all plasmids. We learned a lesson from this and tried to avoid six-fragment Gibson assembly in subsequent experiments.

然而,由于我们需要通过六片段Gibson组装构建质粒，这很困难且成功率低。同时，实验室需要装修，这导致实验时间不足，所以我们最终无法完成全部质粒的构建。我们从中吸取了教训，在后续实验中尽量避免六片段Gibson组装。

(URA,pSensor,independent expression)pSensor-target_Chk1-Chk1
(URA,pSensor,independent expression)pSensor-target_Chk1-Chk1s
(URA,pSensor,independent expression)pSensor-target_Chk1s-Chk1
(URA,pSensor,independent expression)pSensor-target_Chk1s-Chk1s

(URA,pSensor，独立表达)pSensor-target_Chk1-Chk1
(URA,pSensor，独立表达)pSensor-target_Chk1-Chk1s
(URA,pSensor，独立表达)pSensor-target_Chk1s-Chk1
(URA,pSensor，独立表达)pSensor-target_Chk1s-Chk1s

1. pSensor-target_Chk1-Chk1

1. pSensor-target_Chk1-Chk1

Step1. Design of plasmid:

Step1. 质粒设计：

The function of gRNA is to simulate the transcribed mRNA. According to our design, the transcribed RNA Chk1 can bind to the transcribed ogRNA_Chk1 to produce an A-C mismatch at the stop codon, and the UAG on the ogRNA will be edited by ADAR to UIG, so that translation continues, and both the red and the green fluorescent gene can be expressed. AmpR is a marker gene used for E.coli screening and URA3 is a marker gene used for yeast screening.

gRNA的功能是模拟转录的mRNA。根据我们的设计，转录的RNA Chk1可以与转录的ogRNA_Chk1结合，在终止密码子处产生A-C错配，ogRNA上的UAG将被ADAR编辑为UIG，从而使翻译继续进行，红色和绿色荧光基因都能表达。AmpR是用于大肠杆菌筛选的标记基因，URA3是用于酵母筛选的标记基因。

Step2. Electrophoretic results:

Step2. 电泳结果：

We randomly picked 8 single colonies on the culture medium for colony PCR. KK group in the following figure represents the results of the construction of pSensor-target_Chk1-Chk1.

我们在培养基上随机挑选了8个单菌落进行菌落PCR。下图中的KK组代表pSensor-target_Chk1-Chk1构建的结果。

The correct band will be 1000bp long.

正确的条带长度应为1000bp。

Step3. Sequencing results:

Step3. 测序结果：

The company's sequencing results show that our construction of pSensor-target_Chk1-Chk1 plasmid is completely right.

公司的测序结果显示，我们构建的pSensor-target_Chk1-Chk1质粒完全正确。

2. pSensor-target_Chk1-Chk1s

2. pSensor-target_Chk1-Chk1s

Step1. Design of plasmid:

Step1. 质粒设计：

This plasmid was designed as a control. According to our design, the transcribed RNA Chk1 can not bind to the transcribed ogRNA_Chk1s to produce an A-C mismatch at the stop codon, and the UAG on the ogRNA will still stop the translation, so that transcription continues, and only the red fluorescent gene can be expressed. AmpR is a marker gene used for E.coli screening and URA3 is a marker gene used for yeast screening.

这个质粒被设计为对照。根据我们的设计，转录的RNA Chk1不能与转录的ogRNA_Chk1s在终止密码子处产生A-C错配，ogRNA上的UAG仍将终止翻译，因此只有红色荧光基因能够表达。AmpR是用于大肠杆菌筛选的标记基因，URA3是用于酵母筛选的标记基因。

Step2. Electrophoretic results:

Step2. 电泳结果：

We randomly picked 8 single colonies on the culture medium for colony PCR. The correct bands of three ports will all be 1000bp long.

我们在培养基上随机挑选了8个单菌落进行菌落PCR。三个端口的正确条带长度都应为1000bp。

Step3. Sequencing results:

Step3. 测序结果：

The company's sequencing results show that our construction of pSensor-target_Chk1-Chk1s plasmid is completely right.

公司的测序结果显示，我们构建的pSensor-target_Chk1-Chk1s质粒完全正确。

3. pSensor-target_Chk1s-Chk1

3. pSensor-target_Chk1s-Chk1

Step1. Design of plasmid:

Step1. 质粒设计：

This plasmid was designed as a control. According to our design, the transcribed RNA Chk1s can not bind to the transcribed ogRNA_Chk1 to produce an A-C mismatch at the stop codon, and the UAG on the ogRNA will still stop the translation, so that transcription continues, and only the red fluorescent gene can be expressed. AmpR is a marker gene used for E.coli screening and URA3 is a marker gene used for yeast screening..

这个质粒被设计为对照。根据我们的设计，转录的RNA Chk1s不能与转录的ogRNA_Chk1在终止密码子处产生A-C错配，ogRNA上的UAG仍将终止翻译，因此只有红色荧光基因能够表达。AmpR是用于大肠杆菌筛选的标记基因，URA3是用于酵母筛选的标记基因。

Step2. Electrophoretic results:

Step2. 电泳结果：

We randomly picked 8 single colonies on the culture medium for colony PCR. SK_Port1 and SK_Port2 group in the following figure represent the results of the construction of pSensor-target_Chk1s-Chk1.

我们在培养基上随机挑选了8个单菌落进行菌落PCR。下图中的SK_Port1和SK_Port2组代表pSensor-target_Chk1s-Chk1构建的结果。

The correct band of port1 will be 1000bp long and the correct band of port2 will be 2900bp. False positive control comes from the template plasmid (pSensor-target_Chk1-Chk1s) with the same identity primers for port2.

端口1的正确条带长度应为1000bp，端口2的正确条带长度应为2900bp。假阳性对照来自具有相同身份引物的模板质粒(pSensor-target_Chk1-Chk1s)用于端口2。

Step3. Sequencing results:

Step3. 测序结果：

The company's sequencing results show that our construction of pSensor-target_Chk1s-Chk1 plasmid is completely right.

公司的测序结果显示，我们构建的pSensor-target_Chk1s-Chk1质粒完全正确。

4. pSensor-target_Chk1s-Chk1s

4. pSensor-target_Chk1s-Chk1s

Step1. Design of plasmid:

Step1. 质粒设计：

The function of gRNA is to simulate the transcribed mRNA. According to our design, the transcribed RNA Chk1s can bind to the transcribed ogRNA_Chk1s to produce an A-C mismatch at the stop codon, and the UAG on the ogRNA will be edited by ADAR to UIG, so that translation continues, and both the red and the green fluorescent gene can be expressed. AmpR is a marker gene used for E.coli screening and URA3 is a marker gene used for yeast screening.

gRNA的功能是模拟转录的mRNA。根据我们的设计，转录的RNA Chk1s可以与转录的ogRNA_Chk1s结合，在终止密码子处产生A-C错配，ogRNA上的UAG将被ADAR编辑为UIG，从而使翻译继续进行，红色和绿色荧光基因都能表达。AmpR是用于大肠杆菌筛选的标记基因，URA3是用于酵母筛选的标记基因。

Step2. Electrophoretic results:

Step2. 电泳结果：

We randomly picked 8 single colonies on the culture medium for colony PCR. SS group in the following figure represents the results of the construction of pSensor-target_Chk1s-Chk1s. The correct band will be 2900bp long.

我们在培养基上随机挑选了8个单菌落进行菌落PCR。下图中的SS组代表pSensor-target_Chk1s-Chk1s构建的结果。正确的条带长度应为2900bp。

Step3. Sequencing results:

Step3. 测序结果：

The company's sequencing results show that our construction of pSensor-target_Chk1s-Chk1s plasmid is completely right.

公司的测序结果显示，我们构建的pSensor-target_Chk1s-Chk1s质粒完全正确。

During the culture process of yeast transformation, most of our strains contain the correct plasmids. Following figures show the results. Begin with the Marker, each two bands represent a single clone Yeast. First band stands for plasmids pSensor-target, and the second one stands for corresponding ADAR1 or ADAR2. We tested the second band for confirming the chassis microbes we used for Yeast transformation keep the right ADAR we transformed previously.

在酵母转化的培养过程中，我们的大多数菌株都含有正确的质粒。以下图片显示了结果。从Marker开始，每两个条带代表一个单克隆酵母。第一个条带代表pSensor-target质粒，第二个条带代表相应的ADAR1或ADAR2。我们测试了第二个条带，以确认我们用于酵母转化的底盘微生物保留了我们之前转化的正确ADAR。

To evaluate the success of the system we designed, we assessed the fluorescence intensity of each group. Initially, we utilized LSCM for detection.

By measuring the fluorescence intensity and calculating the ratio of eGFP to mScarlet, we can determine the editing efficiency of the ADARs.

为了评估我们设计的系统的成功性，我们评估了每组的荧光强度。最初，我们使用LSCM进行检测。

通过测量荧光强度并计算eGFP与mScarlet的比率，我们可以确定ADAR的编辑效率。

The ratio of two fluorescences showed some results out of our expectations. Clear fluorescences gave another proof of the successfully use of our system, though the ratio datas are not that persuasive. Flow cytometry analysis results will give us more evidences.

两种荧光的比率显示了一些出乎我们意料的结果。清晰的荧光为我们系统的成功使用提供了另一个证明，尽管比率数据不那么具有说服力，流式细胞术分析结果将为我们提供更多证据。

We got nice datas from Flow cytometry analysis, though the results were not exactly as what we expected. The gates we had drawn to distinguish different cell signals are shown in Fig91.

我们从流式细胞术分析中获得了不错的数据，尽管结果与我们的预期有些不同。我们用于区分不同细胞信号的门限设置如图91所示。

Also, to show our results more clearly, we drew cross lines to divide each picture into four parts( these cross gates are not how we got our number datas, just drew to make readers understand easier). And we obtained the number datas using the gates in Fig91.

为了更清晰地展示我们的结果，我们在每张图片上画了十字线，将其分成四个部分（这些十字门限不是我们获取数据的方式，只是为了让读者更容易理解）。我们使用图91中的门限获得了数值数据。

Then we processed the datas into bar chart following the formula: Edit Efficiency = $\frac{100 \times \text{mScarlet} \& \text{eGFP}}{\text{mScarlet}} \%$

然后我们按照以下公式将数据处理成条形图：编辑效率 = $\frac{100 \times \text{mScarlet} \& \text{eGFP}}{\text{mScarlet}} \%$

According to the datas, we came to some conclusions:

根据这些数据，我们得出了一些结论：

1. TA2 has the higher edit efficiency at about 80-95%, but this causes no evidence of selectivity. So we couldn't evaluate whether our sensor function well or not according to the editing effect cause by TA2.

2. The extremely high efficiency of TA2 in yeast shows the potential of becoming a powerful gene edit tool in yeast.

3. TA1 has the edit efficiency >15% according to the datas from our Optimization group, which is significantly higher than the stop codon readthrough or off target effect (edit efficiency is around 5%). So we could infer that TA1 has a strong selective edit effect for pSensor-Chk1 with Target-Chk1. But TA1 only has the edit effect on pSensor-target_Chk1-Chk1, that was beyond our expectations.

4. Design of Sensor is important for the selectivity of ADAR. After the experiment, we put the sequences into IntaRNA and predict the binding energy and binding area of sensor and its corresponding target. According to the results differences, we inferred that the sensor might not have more than one potential binding area and the base around the A-C mismatch might be strictly complementary pairing (not pairing like CAU-AUC).

1. TA2的编辑效率较高，约为80-95%，但这并不能证明其选择性。因此，我们无法根据TA2引起的编辑效果评估我们的传感器是否运作良好。

2. TA2在酵母中极高的效率显示了它成为酵母中强大的基因编辑工具的潜力。

3. 根据我们优化组的数据，TA1的编辑效率>15%，显著高于终止密码子读穿或非特异性效应（编辑效率约为5%）。因此，我们可以推断TA1对带有Target-Chk1的pSensor-Chk1具有强烈的选择性编辑效果。但TA1仅对pSensor-target_Chk1-Chk1有编辑效果，这超出了我们的预期。

4. 传感器的设计对ADAR的选择性很重要。实验后，我们将序列输入IntaRNA并预测传感器及其相应靶标的结合能量和结合区域。根据结果差异，我们推断传感器可能没有多个潜在结合区域，A-C错配周围的碱基可能需要严格的互补配对（而不是像CAU-AUC这样的配对）。

After we got the experiment datas, we analyzed them carefully. We found there are several problems we need to address.

在获得实验数据后，我们仔细分析了它们。我们发现有几个问题需要解决：

1. Whether PDC1-ADAR1 and PDC1-ADAR_MCP will work as the same as TEF-ADAR1.

2. Why group TA1-SS do not have the same results as TA1-KK as we expected.

3. Why TEF-ADAR_MCP has no selectivity for all four plasmids.

4. Whether the CAU-AUC sequence cause the decreasing edit efficiency of ADAR.

1. PDC1-ADAR1和PDC1-ADAR_MCP是否会与TEF-ADAR1的作用相同。

2. 为什么TA1-SS组没有像我们预期的那样与TA1-KK组产生相同的结果。

3. 为什么TEF-ADAR_MCP对所有四种质粒都没有选择性。

4. CAU-AUC序列是否导致ADAR的编辑效率降低。

Due to the time limited, further experiments will be designed later.

由于时间有限，将在后续设计进一步的实验。

Due to these issues that we hadn't addressed, further experiments are needed. We will look for a promoter whose efficiency is higher than PDC1 and lower than TEF to find a better promoter for ADAR_MCP. Also we will design a better sequence for gRNA to improve its A-C mismatch area and specificity. And there are a lot of things that we need to explore, including find whether TEF-ADAR_MCP could edit without target gene(some of our experiments could explain this, but we need more evidence to confirm )

由于这些尚未解决的问题，需要进行更多实验。我们将寻找效率高于PDC1但低于TEF的启动子，以找到更适合ADAR_MCP的启动子。我们还将为gRNA设计更好的序列，以改善其A-C错配区域和特异性。此外，还有许多需要探索的内容，包括确定TEF-ADAR_MCP是否可以在没有靶基因的情况下进行编辑（我们的一些实验可以解释这一点，但我们需要更多证据来确认）。

Both ADAR1 and ADAR_MCP demonstrated editing ability in yeast, but the underlying mechanisms are complex. We observed some positive results regarding selective ability, though the outcomes were somewhat unexpected.. More experiments are needed. We are also eager to find out more potential of this monitoring system.

ADAR1和ADAR_MCP在酵母中都表现出了编辑能力，但其underlying机制比较复杂。我们观察到了一些关于选择能力的积极结果，尽管结果有些出乎意料。需要进行更多实验。我们也渴望发现这个监测系统的更多潜力。

[1] Martin Mann, Patrick R. Wright, and Rolf Backofen IntaRNA 2.0: enhanced and customizable prediction of RNA–RNA interactions Nucleic Acids Research, 2017, 45 (W1), W435–W439.
[2] Patrick R. Wright, Jens Georg, Martin Mann, Dragos A. Sorescu, Andreas S. Richter, Steffen Lott, Robert Kleinkauf, Wolfgang R. Hess, and Rolf Backofen CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains Nucleic Acids Research, 2014, 42 (W1), W119-W123.
[3] Anke Busch, Andreas S. Richter, and Rolf Backofen IntaRNA: efficient prediction of bacterial sRNA targets incorporating target site accessibility and seed regions Bioinformatics, 2008, 24 (24), 2849-56.
[4] Martin Raden, Syed M Ali, Omer S Alkhnbashi, Anke Busch, Fabrizio Costa, Jason A Davis, Florian Eggenhofer, Rick Gelhausen, Jens Georg, Steffen Heyne, Michael Hiller, Kousik Kundu, Robert Kleinkauf, Steffen C Lott, Mostafa M Mohamed, Alexander Mattheis, Milad Miladi, Andreas S Richter, Sebastian Will, Joachim Wolff, Patrick R Wright, and Rolf Backofen Freiburg RNA tools: a central online resource for RNA-focused research and teaching Nucleic Acids Research, 46(W1), W25-W29, 2018.

[1] Martin Mann, Patrick R. Wright, and Rolf Backofen IntaRNA 2.0: enhanced and customizable prediction of RNA–RNA interactions Nucleic Acids Research, 2017, 45 (W1), W435–W439.
[2] Patrick R. Wright, Jens Georg, Martin Mann, Dragos A. Sorescu, Andreas S. Richter, Steffen Lott, Robert Kleinkauf, Wolfgang R. Hess, and Rolf Backofen CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains Nucleic Acids Research, 2014, 42 (W1), W119-W123.
[3] Anke Busch, Andreas S. Richter, and Rolf Backofen IntaRNA: efficient prediction of bacterial sRNA targets incorporating target site accessibility and seed regions Bioinformatics, 2008, 24 (24), 2849-56.
[4] Martin Raden, Syed M Ali, Omer S Alkhnbashi, Anke Busch, Fabrizio Costa, Jason A Davis, Florian Eggenhofer, Rick Gelhausen, Jens Georg, Steffen Heyne, Michael Hiller, Kousik Kundu, Robert Kleinkauf, Steffen C Lott, Mostafa M Mohamed, Alexander Mattheis, Milad Miladi, Andreas S Richter, Sebastian Will, Joachim Wolff, Patrick R Wright, and Rolf Backofen Freiburg RNA tools: a central online resource for RNA-focused research and teaching Nucleic Acids Research, 46(W1), W25-W29, 2018.