RNA contains information of life. Understanding RNA can help us better understand metabolism, bio-factory, disease and so on. We developed RNAssay, a molecular tool that are hopeful to carry out in situ RNA sensing, which can achieve different functions with different collocation of basic gene, reporter gene and ogRNA. According to our investigation and experiment, we propose at least three possible applications for our project: in situ detection of multigene expression, strain security system and in vivo dynamic monitoring of splice variants, corresponding to three different types of end users and different requirements.

RNA包含生命的信息。理解RNA可以帮助我们更好地了解代谢、生物工厂、疾病等。我们开发了RNAssay，这是一种有望实现原位RNA感应的分子工具，通过基本基因、报告基因和ogRNA的不同搭配可以实现不同的功能。根据我们的调查和实验，我们为我们的项目提出至少三种可能的应用：多基因表达的原位检测、防盗门和剪接异构体的胞内动态监测，这些对应着三种不同类型的使用者和需求。

The end users of in situ detection of multigene expression are researchers in lab and enterprise managers of cell-factory. Researchers can use RNAssay as qPCR or other tools to analyze the RNA level of target gene, guiding further experiment design. And enterprise managers can use RNAssay to analyze and predict the production of its factory in real time. To make it as helpful as qPCR, it should meet the requirement of high precision, high accuracy and high sensitivity and low detection limit.

原位多基因表达检测的使用者是实验室研究人员和细胞工厂的企业管理者。研究人员可以像使用qPCR或其他工具一样使用RNAssay来分析目标基因的RNA水平，从而指导进一步的实验设计。而企业管理者可以使用RNAssay实时分析和预测其工厂的产量。为了使其像qPCR一样有用，它应该满足高精度、高准确度、高灵敏度和低检测限的要求。

The end users of strain security system are mainly enterprise managers of cell-factory and culture preservation center (eg: China General Microbiological Culture Collection Center). Applying the security system can help them better protect the key culture, which is good for both profit and reputation. A good strain security system should meet the requirement of high stability and high cryptographic strength.

工业菌株防盗门的最终用户主要是细胞工厂和菌种保藏中心的企业管理者（例如：中国普通微生物菌种保藏管理中心）。应用这种安全系统可以帮助他们更好地保护关键菌种，这对利润和声誉都有好处。一个良好的菌株安全系统应该满足高稳定性和高加密强度的要求。

The end users of in vivo dynamic monitoring of splice variants are researchers in lab and enterprise managers of disease detection. Utilizing our system can better distinguish different splice variant in the form of mature mRNA. The key requirement of the system should be high resolution ratio and general applicability.

剪接异构体的胞内动态监测的最终用户是实验室的研究人员和疾病检测的企业管理者。利用我们的系统可以更好地以成熟mRNA的形式区分不同的剪接异构体。该系统的关键要求应该是高分辨率和普遍适用性。

Here, we propose two possible implementation plan: standardized plan and personalized plan.

在此，我们提出两种可能的实施方案：标准化方案和个性化方案。

For standardized plan, we learn from the case of protein tags like FLAG. We'll provide standardized sensor with specific ogRNA that target to a special RNA sequence, namely, target sequence. When users are designing their plasmid, they can choose to attach one of the standardized tag sequences (target sequence + 2A peptide sequence) to the 5' end of the gene that require in situ detection. And chassis microorganisms are also provided, which has been transformed with optimized adar and sensor that are inducibly expressed. Researchers need no extra operation to make their gene visible in RNA level, with only one transformation of the target gene.

对于标准化方案，我们借鉴了蛋白质标签（如FLAG）的做法。我们将提供标准化的传感器，其中包含特定的ogRNA，用于靶向特殊的RNA序列，即目标序列。当用户设计他们的质粒时，他们可以选择将其中一个标准化的标签序列（目标序列 + 2A肽序列）附加到需要原位检测的基因的5'端。同时，我们还提供经过改造的底盘微生物，这些微生物已经转化了可诱导表达的优化adar和传感器。研究人员无需额外操作即可在RNA水平上使他们的基因可见，只需进行一次目标基因的转化即可。

For personalized plan, one can design customized sensor, which can be achieved automatically with RADARS Guide Design Tool. Unlike the standardized plan, the chassis microorganisms provided will only carry adar gene, since the sensor is customized. So researchers will need one extra operation to transform the sensor, unless they load the sensor and their target gene onto a single plasmid.

对于个性化方案，可以设计定制的传感器，这可以通过Guide设计工具自动实现。与标准化方案不同，提供的底盘微生物只会携带adar基因，因为传感器是定制的。因此，研究人员需要额外的一步操作来转化传感器，除非他们将传感器和目标基因装载到同一个质粒上。

Once the plasmids are successfully transformed, researchers are able to characterize the target gene by ELISA reader, fluorescence microscope or flow cytometer. And for bio-manufacturing enterprises, they can periodically fetch some sample from the fermentation tank to carry out further characterization.

一旦质粒成功转化，研究人员就能够通过ELISA读数器、荧光显微镜或流式细胞仪来表征目标基因。对于生物制造企业而言，他们可以定期从发酵罐中取样，进行进一步的表征分析。

To achieve the aforementioned implementation plan, some further study need to be done.

为了实现上述实施计划，还需要做一些进一步的研究。

1.Reduce cytotoxicity of ADAR

1.减轻ADAR的细胞毒性

According to our experiment result, we discover that the ADAR enzyme may somehow suppress the growth of yeast. So extra modification to ADAR are necessary. One possible mechanism for the cytotoxicity of ADAR2 may be that ADAR2 edits the mRNA of the transcription initiation protein Bdf2p, inhibiting cell growth. And overexpression of the BDF2 gene can restore growth$^1$.

根据我们的实验结果，我们发现ADAR酶可能以某种方式抑制了酵母的生长。因此，对adar进行额外的修改是必要的。ADAR2产生细胞毒性的一种可能机制是，ADAR2编辑转录起始蛋白Bdf2p的mRNA，从而抑制细胞生长。而过表达BDF2基因可以恢复生长$^1$。

1.Construct standard sensor and standard chassis microorganism

1.构建标准传感器和标准底盘微生物

2.Optimize the sensor design

2.优化传感器设计

Since a single gene can have multiple sensor options, a strategy to select the best sensor can be helpful. We have tried to use our dry lab model to solve the problem but apparently there are still long way to go.

由于单个基因可能有多个传感器选项，选择最佳传感器的策略可能会很有帮助。我们曾尝试使用我们的干实验室模型来解决这个问题，但依然任重道远。

To apply our strain security system, end users will need to carry out two more transformation to load this system (ADAR+Sensor) into their strain. Once the strain are loaded with our security system, it can only be thawed under certain environmental stress such as starvation and high temperature. And classical method like microbial replica plating can be useful for screening successful strains.

为了应用我们的防盗门，用户需要进行两次额外的转化以将该系统（ADAR+传感器）转入到他们的菌株中。一旦菌株被转入了我们的防盗门，它只能在特定的环境压力下解冻，例如饥饿状态和高温环境。

To be honest, our strain security system still lacks a lot to be truly implemented.

诚然，我们的压力安全系统要想真正实施，还有很多不足之处。

1.Expanding the scope of protection

1.扩大防盗范围

During the human practice with EVOLYZER, we noticed that most strain stealing happens in the factory, or just right beside the fermentation tank. So our system can hardly eradicate strain stealing. We have to admit that its really hard to construct a truly useful strain security system. We believe a periodically suicide system could be helpful, and epigenetics can be utilized. One possible design we came up utilize the methylase and promoter methylation in human cell. As shown in Fig 3, the new design of strain security system contains four main part, mostly same as the previous design. One key different is the reporter gene of sensor has changed into a special methylase, which can methylate a specific promoter, thus inhibit the downstream expression of sensor, which consists of a suicide gene. And all the above situation will only happen with environmental stress (starvation/high temperature), otherwise the suicide gene will express normally and kill the cell. The main advantage of this system is that promoter methylation decreases with cell division, so once methylation only last for certain times. In another word, strain with this security system will need to treat with environmental stress periodically to survive.

在人类文明实践与EVOLYZER的交流中，我们了解到大多数菌株盗窃发生在工厂内，或者就在发酵罐旁边。因此，我们的系统很难根除菌株盗窃。我们必须承认，构建一个真正有用的菌株安全系统难度较大。但我们认为，一个周期性的自我毁灭系统可能会有帮助，并且可以利用表观遗传学。我们想出了一个可能的设计，即利用人类细胞中的甲基化酶和启动子甲基化。如图3所示，新的菌株安全系统设计包含四个主要部分，这与之前的设计理念大致相同。一个关键的不同之处在于，传感器的报告基因变为了一种特殊的甲基化酶，它能够甲基化一个特定的启动子，从而抑制传感器下游的表达，这包括了一个自杀基因。而且，上述所有情况只会在环境压力（饥饿/高温）下发生，否则自我毁灭基因将正常表达并杀死细胞。该系统的主要优点是，随着细胞分裂，启动子甲基化会减少，所以甲基化只能持续一定的时间。换句话说，带有这种安全系统的菌株需要定期处理环境压力才能生存。

However, to date, no similar cases have been reported in yeast and no relavant molecular tool has been developed. From the design to truly apply into industry, there's still a long way to go.

然而，迄今为止，在酵母中还没有类似的案例报道，也没有相关的分子工具被开发出来。从设计到真正应用于工业，还有很长的路要走。

It has beem concluded that there are two main types of end users of in vivo dynamic monitoring of splice variants: researchers in lab and enterprise managers.

已经得出结论，胞内剪接异构体动态监测的用户主要有两类：实验室研究人员和企业管理者。

For researchers, it can be simply used in the same way of in vivo detection of multigene expression. The key point should be designing proper sensors that can distinguish different splice variant. For the general applicability, according to our dry lab result, approximately 20% of human splice variants can be distinguished by RNAssay, and our dry lab model will be useful to guide you design your own sensor. Besides fluorescent proteins, the reporter genes can be changed into other genes to achieve multiple functions.

对于研究人员来说，它可以像胞内多基因表达检测一样简单地使用。关键点应该是设计适当的传感器，能够区分不同的剪接异构体。就普遍适用性而言，根据我们的干实验室结果，大约20%的人类剪接异构体可以通过RNAssay进行区分，我们的干实验室模型将有助于指导设计自己的传感器。除了荧光蛋白外，报告基因还可以更改为其他基因以实现多种功能。

For enterprise managers, cooperations can be made with hospitals. Since splice variants are significant cancer signals in some cases. The companies may collect samples from different hospitals and return test result, to help detect the risk of cancer. And different types of samples are needed to diagnose different types of cancer.

对于企业管理者来说，可以与医院建立合作关系。由于在某些情况下，剪接异构体是重要的癌症信号。公司可以从不同的医院收集样本并返回测试结果，以帮助检测癌症风险。而且，诊断不同类型的癌症需要不同类型的样本。

Apart from disease detection, another hopeful application may relate to “next generation organ transplantation", which indicates stem cell reprogramming to culture organs for transplantation, avoiding autoimmune rejection. Tumorigenesis are key challenges in the field of stem cell reprogramming. We can use our RNAssay to capture cancerization signals from splice variants and initiate suicide procedure (Fig 5). And considering safety requirement, cre-loxp recombination system can be combined with RNAssay to remove the sensor, while adar is endogenous (Fig 6).

除了疾病检测之外，另一个有希望的应用可能与"下一代器官移植"有关。这指的是通过干细胞重编程培养用于移植的器官，从而避免自身免疫排斥反应。在干细胞重编程领域，肿瘤发生是一个关键挑战。我们可以使用我们的RNAssay来捕获来自剪接异构体的癌变信号，并启动自杀程序（图4）。考虑到安全要求，Cre-loxP重组系统可以与RNAssay结合使用以移除传感器，而ADAR是内源性的（图6）。

1.Apply the system to human cell

1.将该系统应用于人体细胞

We tested the potential for monitoring splice variants in yeast. However, due to the limitation of time and resources, we failed to test the system in human cells. Further work need to be done before its potential is truly demonstrated.

我们测试了在酵母中监测剪接异构体的潜力。然而，由于时间和资源的限制，我们未能在人类细胞中测试该系统。在其潜力真正得到证实之前，还需要进行进一步的工作。

2.Test the limitation of RNAssay

2.检测RNAssay的应用范围

Since there can be different junctions for different splice varient, and CCA sequence may locate in different position beside the junctions. Some questions need to be answered by experiment: what is the longest distance between CCA location and the junction, what is the best position for CCA sequence?

由于不同的剪接异构体可能有不同的连接点，且 CCA 序列可能位于连接点附近的不同位置，一些问题需要通过实验来回答：CCA 位置与连接点之间的最大距离是多少，CCA 序列的最佳位置是什么？

1 Eifler, T., Pokharel, S. & Beal, P. A. RNA-Seq analysis identifies a novel set of editing substrates for human ADAR2 present in Saccharomyces cerevisiae. Biochemistry 52, 7857-7869 (2013).

1 Eifler, T., Pokharel, S. & Beal, P. A. RNA-Seq analysis identifies a novel set of editing substrates for human ADAR2 present in Saccharomyces cerevisiae. Biochemistry 52, 7857-7869 (2013).