To prove the concept, three basic parts were designed for our system: ADAR, target transcript and sensor for that target (Fig.1).

为了证明这个概念，我们为系统设计了三个基本部分：ADAR、目标转录本和针对该目标的传感器

ADARs (Adenosine Deaminases Acting on RNA) are a family of enzymes that catalyze the conversion of adenosine to inosine in double-stranded RNA. This RNA editing process alters the nucleotide sequence of RNA transcripts, potentially changing the coding sequence and affecting gene expression. The current experimental report has identified three main families of ADAR proteins, each of which has homologous sequences present in various mammals.

ADARs（作用于RNA的腺苷脱氨酶）是一类催化双链RNA中腺苷转化为肌苷的酶家族。这种RNA编辑过程改变了RNA转录本的核苷酸序列，可能会改变编码序列并影响基因表达。当前的实验报告已经确定了三个主要的ADAR蛋白家族，每个家族在各种哺乳动物中都有同源序列存在。

As yeasts don't have endogenous ADAR, we need to introduce exogenous ADAR and make sure it functions well in yeast. The most representative ADAR proteins are ADAR1 (P110), ADAR1 (P150), ADAR2, and ADAR3.

由于酵母没有内源性ADAR，我们需要引入外源ADAR并确保它在酵母中功能良好。最具代表性的ADAR蛋白是ADAR1（P110）、ADAR1（P150）、ADAR2和ADAR3。

Based on research situation, migratory capability, uniform expression quality, programmability, subcellular localization, and specificity (off-target rate), several ADARs were evaluated. The information for ADAR3 has not been very clear until now; ADAR1_p110 is mainly located in the nucleolus and it might not be suitable for our system for matural RNA need to be edited in cytoplasma. Also, testing at least two types of ADAR is a better choice and it was reported that both ADAR1 and ADAR2 have catalysis activity in mammal cells$^{1}$.

基于研究现状、迁移能力、表达质量的一致性、可编程性、亚细胞定位和特异性（脱靶率），我们对几种ADAR进行了评估。目前为止，关于ADAR3的信息还不是很清楚；ADAR1_p110主要位于核仁中，可能不适合我们的系统，因为成熟的RNA需要在细胞质中进行编辑。此外，测试至少两种类型的ADAR是更好的选择，有报道表明ADAR1和ADAR2在哺乳动物细胞中都具有催化活性$^{1}$。

Finally, ADAR1_p150 and ADAR2(DD)E488Q–MCP were selected and expressed through homologous recombination or independent expression in yeast respectively. As reported, ADAR1_p150 is an isoform of ADAR1 with higher edit efficiency and MCP domain help ADAR2 specifically be recruited by MS2 structure$^{2,3}$. These two forms of ADAR have the highest potential for our system. The ADAR with the highest editing efficiency will be choosed for consequent experiments.

最终，我们选择了ADAR1_p150和ADAR2(DD)E488Q–MCP，并分别通过同源重组或独立表达的方式在酵母中表达。据报道，ADAR1_p150是ADAR1的一种异构体，具有更高的编辑效率，而MCP结构域则帮助ADAR2特异性地被MS2结构招募$^{2,3}$。这两种形式的ADAR对我们的系统来说具有最高的潜力。我们将选择编辑效率最高的ADAR进行后续实验。

The sensor is the device which is programmable to sense target RNA, which can be divided into 5 parts: upstream gene, 2A peptide 1, organized guide RNA(ogRNA), 2A peptide 2 and downstream gene (Fig.1). Here, we called upstream gene as (basic gene) and downstream gene as (reporter gene). To prove the concept, firstly EGFP was chosen as our basic gene, and EBFP as our reporter gene. Only green fluorescence was expected to be seen when off target and both green and blue fluorescence were expected to be seen when with target.

传感器是可编程以感知目标RNA的部分，它可以分为5个部分：上游基因、2A肽1、组织化引导RNA（ogRNA）、2A肽2和下游基因（图1）。在这里，我们将上游基因称为（基础基因），下游基因称为（报告基因）。为了证明这个概念，我们首先选择EGFP作为基础基因，EBFP作为报告基因。预期在无目标情况下只能观察到绿色荧光，而在有目标存在时，预期可以同时观察到绿色和蓝色荧光。

ogRNA are complementary sequence of the target, which function as compass for the sensor. The ACC codon and the sequence near ACC in the target transcript are the target area we may consider for designing our Sensor. Because there might be many ACC codons in target gene and when designing the Sensor, extract stop codon must no exist in it. So, it's hard for us to design and choose the most suitable sensor sequence only by experience. The sequence between EGFP and EBFP was designed by a program derived from our foundational references (Fig.1), details are in our dry lab.

ogRNA是目标序列的互补序列，它作为传感器的指南。目标转录本中的ACC密码子及其周围序列是我们在设计传感器时可能考虑的目标区域。因为目标基因中可能存在多个ACC密码子，且在设计传感器时，其中不能存在提取终止密码子。因此，仅凭经验来设计和选择最合适的传感器序列对我们来说是困难的。EGFP和EBFP之间的序列是通过一个源自我们基础参考文献的程序设计的（图1），详细信息在我们的干实验室部分。

There must be a UGA stop codon in the middle of the sensor. According to our foundational references, adding MS2 around the stop codon could enhance the specificity and editing efficiency of ADAR. This is because MS2 could form a loop structure and there are one or more motifs of ADAR could recognize MS2 loop. So we imitated the design of our foundational references by adding 4 MS2 sequences, each side of the stop codon has two.

传感器的中间必须有一个UGA终止密码子。根据我们的基础参考文献，在终止密码子周围添加MS2序列可以提高ADAR的特异性和编辑效率。这是因为MS2可以形成一个环状结构，而且ADAR有一个或多个模体可以识别MS2环。因此，我们模仿了基础参考文献的设计，添加了4个MS2序列，终止密码子的每侧各有两个。

When the stop codon was edited to non-stop codon, protein EGFP will connect with EBFP, and this might cause functional loss. To make EGFP and EBFP work sucessfully, there must be two 2A peptides, one lies after EGFP and another before the EBFP. The 2A peptide induces a "ribosomal skipping" mechanism during translation$^{4}$. When the ribosome translates the 2A peptide sequence, it releases the formation of a peptide bond at a specific position but does not terminate the entire translation process. As a result, the downstream protein can continue to be translated, but it is linked to the upstream protein through non-covalent bonds, allowing the final two proteins to exist independently within the cell. Therefore, both EGFP and EBFP could function well in the specific situation we provide.

当终止密码子被编辑成非终止密码子时，EGFP蛋白将与EBFP连接，这可能导致功能丧失。为了使EGFP和EBFP能够成功工作，必须有两个2A肽，一个位于EGFP之后，另一个位于EBFP之前。2A肽在翻译过程中引发一种"核糖体跳跃"机制$^{4}$。当核糖体翻译2A肽序列时，它会在特定位置释放肽键的形成，但不会终止整个翻译过程。因此，下游蛋白质可以继续被翻译，但它通过非共价键与上游蛋白质相连，使得最终两种蛋白质能够在细胞内独立存在。因此，在我们提供的特定情况下，EGFP和EBFP都能正常发挥功能。

Target gene was which we want to detect. Both EBFP and EGFP were expected to be seen when target exists and EGFP but no EBFP without target.

我们想要检测的是目标基因。当靶标存在时，预计可以看到 EBFP 和 EGFP，而当没有靶标时，预计可以看到 EGFP 但没有 EBFP。

Normally, a suitable gene must be firstly chose as our target. However, choosing a gene of yeast involves the unknown problem of whether its function will be disturb by our system; choosing a gene of other species involves the unknown problem of how well it could express in yeast.

通常，我们必须首先选择一个合适的基因作为我们的目标。然而，选择酵母的基因涉及到一个未知问题，即其功能是否会被我们的系统干扰；选择其他物种的基因则涉及到另一个未知问题，即它在酵母中的表达效果如何。

To quickly finish our proof of concept as how well ADAR could function in yeast remained unknown, considering too many things might not be a good choice. So we simplified the design of the sensor and target. After EGFP and EBFP were connected with a sequence of 2A peptide(E2A), a 179bp sequence was cloned from the connection sectiont of EGFP and EBFP, with the codon AUC pairing with stop codon UAG for EGFP in the center of this sequence. This sequence was named as gRNA. Then gRNA was inserted into the same plasmid. At last, codon AUC(pairing with UAG) was mutated to ACC to create a mismatch.

为了快速完成我们的概念验证，因为 ADAR 在酵母中的功能效果仍不清楚，考虑太多因素可能不是一个好选择。所以我们简化了传感器和靶标的设计。在 EGFP 和 EBFP 通过一段 2A 肽（E2A）序列连接后，我们从 EGFP 和 EBFP 的连接部分克隆了一段 179bp 的序列，其中 EGFP 的终止密码子 UAG 与密码子 AUC 配对，位于这个序列的中心。这个序列被命名为 gRNA。然后，gRNA 被插入到同一个质粒中。最后，将配对 UAG 的密码子 AUC 突变为 ACC，创造了一个错配。

After we finished our basic design, we divided our team to three small groups to do the consequent exploration. The first group are going to optimize our system and enhance the editing efficiency; second one will go into the application of Engineering Strain Security System; last will go into the application ofin vivo dynamic monitoring of splice variants.

完成基本设计后，我们将团队分成三个小组进行后续探索。第一组将致力于优化我们的系统并提高编辑效率；第二组将深入研究工程菌株安全系统的应用；最后一组将专注于体内剪接变体动态监测的应用。

In our efforts to enhance the editing activity of ADAR, we replaced the promoter with a stronger one, specifically TEF1, which is one of the strongest promoters in yeast $^5$​.

在我们努力提高 ADAR 编辑活性的过程中，我们用一个更强的启动子TEF1替换了原有的启动子，这是酵母中最强的启动子之一$^5$。

Additionally, we find the eBFP seems weak for our sensor so we redesigned our reporter system, where the basic gene is m-Scarlet and the reporter gene is eGFP. mScarlet is a monomeric synthetic RFP that was recently shown to outperform other RFPs in terms of brightness of the fluorescent signal in yeast$^6$​.

此外，我们发现 eBFP 对我们的传感器来说似乎较弱，所以我们重新设计了我们的报告系统，其中基础基因是 m-Scarlet，报告基因是 eGFP。mScarlet 是一种单体合成红色荧光蛋白，最近的研究表明，在酵母中它的荧光信号亮度优于其他红色荧光蛋白$^6$。

Furthermore, we investigated the influence of the number of MS2 sequences on ADAR editing activity. To this end, we designed three guide RNA (ogRNA) sequences, each containing 0, 2, or 4 MS2 sequences, respectively, while keeping other experimental conditions constant.

此外，我们研究了 MS2 序列数量对 ADAR 编辑活性的影响。为此，我们设计了三种引导 RNA（ogRNA）序列，分别包含 0、2 或 4 个 MS2 序列，同时保持其他实验条件不变。

HSP26

HSP26

Heat shock protein 26(HSP26) encodes a cytosolic member of the small heat shock protein (sHSP) family, which acts as a molecular chaperone$^7$.

热休克蛋白 26（HSP26）编码一种细胞质中的小热休克蛋白（sHSP）家族成员，它作为分子伴侣发挥作用$^7$。

sHSPs bind to and prevent unfolded substrate proteins from irreversibly forming large protein aggregates. The bound substrate proteins can eventually be released and refolded either spontaneously or with the assistance of chaperones$^8$

小热休克蛋白（sHSPs）能与未折叠的底物蛋白结合，防止它们不可逆地形成大型蛋白质聚集体。结合的底物蛋白最终可以被释放，并自发地或在伴侣蛋白的协助下重新折叠$^8$。

HSP26 transcripts are undetectable in unstressed cells but are strongly induced by heat shock, salt shock, cell cycle arrest, nitrogen starvation, carbon starvation, oxidative stress, and low pH$^{8,9,10}$. Under these stress conditions, HSP26 expression is upregulated by the transcription factors Hsf1p and Msn2p/Msn4p, which bind to the heat shock elements and stress response elements in the HSP26 promoter, respectively$^{10}$, which made it a suitable target for our security system.

在未受应激的细胞中，HSP26 转录本是无法检测到的，但在热休克、盐休克、细胞周期阻滞、氮饥饿、碳饥饿、氧化应激和低 pH 条件下会被强烈诱导$^{8,9,10}$。在这些应激条件下，HSP26 的表达被转录因子 Hsf1p 和 Msn2p/Msn4p 上调，这些转录因子分别结合到 HSP26 启动子中的热休克元件和应激响应元件上$^{10}$，这使其成为我们安全系统的合适靶标。

When fermented at 40°C for 6 hours, HSP26 expression levels are upregulated by 166-fold compared to those at 30°C$^{11}$​. This indicates that HSP26 is significantly upregulated under high-temperature conditions to protect cells from heat damage.

当在40°C下发酵6小时，HSP26的表达水平比在30°C下上调了166倍$^{11}$。这表明HSP26在高温条件下显著上调，以保护细胞免受热损伤。

GLC3

GLC3

GLC3 is the gene encoding 1,4-α-glucan-branching enzyme (EC: 2.4.1.18) in Saccharomyces cerevisiae. The gene is located at V:133,120 - 135,234 and is involved in the glycogen biosynthesis process$^{12}$. It adds branches to glycogen molecules$^{13}$. GLC3 mRNA begins to accumulate when environmental glucose decreases to approximately 50% and peaks when environmental glucose is depleted, similar to other glycogen metabolism genes$^{14}$.

GLC3是酿酒酵母中编码1,4-α-葡聚糖支链酶（EC: 2.4.1.18）的基因。该基因位于V:133,120 - 135,234，参与糖原生物合成过程$^{12}$。它的作用是给糖原分子添加支链$^{13}$。当环境中的葡萄糖降低到约50%时，GLC3 mRNA开始积累，并在环境中的葡萄糖耗尽时达到峰值，这与其他糖原代谢基因的表现类似$^{14}$。

GSDMD

GSDMD

Gasdermin-N is a protein that induces pyroptosis in cells, with its domain divided into N-terminal and C-terminal regions. The N-terminal region possesses inherent pyroptosis-inducing activity and is generally believed to aggregate on the cell membrane to form pores, leading to cell pyroptosis. The C-terminal region can inhibit the activity of the N-terminal region; only when the N-terminal and C-terminal regions are separated can pyroptosis be triggered$^{15}$.

Gasdermin-N是一种在细胞中诱导焦亡的蛋白质，其结构域分为N端和C端区域。N端区域具有固有的焦亡诱导活性，通常被认为会在细胞膜上聚集形成孔道，导致细胞焦亡。C端区域可以抑制N端区域的活性；只有当N端和C端区域分离时，才能触发焦亡$^{15}$。

We decide to let yeast cell express the N-terminal domain of GSDMD to achieve its suicide. Experiments have demonstrated that introducing the N-terminal region of GSDMD into yeast cells severely affects their growth$^{16}$​.

我们决定让酵母细胞表达GSDMD的N端结构域以实现其自杀。实验已经证明，将GSDMD的N端区域引入酵母细胞会严重影响其生长$^{16}$。

BAX

BAX

The apoptosis regulator BAX plays a role in the mitochondrial apoptosis pathway$^{17}$. Under normal conditions, BAX is largely cytosolic due to continuous retrotranslocation from the mitochondria to the cytosol mediated by BCL2L1/Bcl-xL, thereby preventing the accumulation of toxic BAX levels at the mitochondrial outer membrane (MOM)$^{18}$. Under stress conditions, BAX undergoes conformational changes that lead to its translocation to the mitochondrial membrane, resulting in the release of cytochrome c, fragmentation of the interconnected mitochondrial network, and subsequent triggering of apoptosis$^{17}$.

凋亡调节因子BAX在线粒体凋亡途径中发挥作用$^{17}$。在正常情况下，由于BCL2L1/Bcl-xL介导的从线粒体到细胞质的持续逆向转运，BAX主要存在于细胞质中，从而防止在线粒体外膜（MOM）上积累有毒的BAX$^{18}$。在压力条件下，BAX经历构象变化，导致其转移到线粒体膜上，引起细胞色素c的释放、相互连接的线粒体网络的碎裂，并随后触发凋亡$^{17}$。

Although full-length Bax protein can hardly induce apoptosis in yeast$^{19}$, it can be functional with minor modifications (Fig.10). Previous reports indicate that C-terminal c-myc-tagged human Bax is highly effective at killing yeast, and this cell death is accompanied by the release of cytochrome c$^{20,21,22,23}$. Additionally, co-expression of Bcl-xL can completely inhibit this death. BaxRC can lead to 50% rate of cell death after cultivating for 16 h, and the rate increase to 57% for Bax$\Delta$C$^{24}$. In our wet lab, we tried all the three modifications.

尽管全长Bax蛋白几乎无法在酵母中诱导凋亡$^{19}$，但经过微小的修饰后它可以发挥功能（图10）。先前的报告表明，C端带有c-myc标签的人源Bax在杀死酵母方面非常有效，这种细胞死亡伴随着细胞色素c的释放$^{20,21,22,23}$。此外，共表达Bcl-xL可以完全抑制这种死亡。BaxRC在培养16小时后可导致50%的细胞死亡率，而Bax$\Delta$C的死亡率增加到57%$^{24}$。在我们的湿实验室中，我们尝试了这三种修饰。

Based on our basic design , three parts still needed to complete: ADAR, Sensor, and Target.

根据我们的基本设计，还需要完成三个部分: ADAR、Sensor和Target。

For ADAR, the plasmid previously constructed in our basic design could be used again. The only different is that previous promoter PDC1 was replaced by a stronger promoter TEF. Details are provided in the part of optimization of our system.

对于ADAR，我们可以再次使用先前在基本设计中构建的质粒。唯一的区别是之前的PDC1启动子被替换为一个更强的TEF启动子。详细信息在我们系统优化部分提供。

For target, a gene that has two splice isoforms, one of which is associated with the normal phenotype and the other with a disease phenotype is the best choice for our system. Choosing a splice viriant related to diseases was aimed at proove the potential of our system to detect diseases caused by abnormal splicing. Currently, the focus of discoveries on abnormal alternative splicing in disease is concentrated on cancer. So, we focus on genes related to cancer.

对于靶标，最适合我们系统的是具有两种剪接异构体的基因，其中一种与正常表型相关，另一种与疾病表型相关。选择与疾病相关的剪接变体旨在证明我们的系统能够检测由异常剪接引起的疾病的潜力。目前，关于疾病中异常选择性剪接的发现主要集中在癌症上。因此，我们专注于与癌症相关的基因。

After looking for target genes through many references, one ideal target genes has been screened out. We finally chose gene Chk1( Checkpoint kinase 1). Chk1 has an alternatively spliced variant, Chk1-s, which skips exon 3 at the N-terminus. Chk1-s inhibits Chk1. In the normal cell cycle, Chk1-s interacts with Chk1, promoting the S-G2/M phase transition. When DNA damage occurs, phosphorylated Chk1 disrupts the Chk1-Chk1-s interaction, activating Chk1 to halt the cell cycle and promote DNA repair. Chk1-s and Chk1 expression levels are higher in fetal and cancer tissues than in normal tissues$^{25}$.

在通过大量参考文献寻找靶基因后，我们筛选出了一个理想的靶基因。我们最终选择了Chk1（检查点激酶1）基因。Chk1有一个选择性剪接变体，称为Chk1-s，它在N端跳过了外显子3。Chk1-s抑制Chk1。在正常的细胞周期中，Chk1-s与Chk1相互作用，促进S-G2/M期转换。当DNA损伤发生时，磷酸化的Chk1打断Chk1-Chk1-s的相互作用，激活Chk1以停止细胞周期并促进DNA修复。Chk1-s和Chk1的表达水平在胎儿和癌症组织中高于正常组织$^{25}$。

As mentioned in description, our system has advantages of Live-cell in situ RNA sensing. The expression levels of Chk1 and Chk1-s exhibit cyclical variations within the cell, and changes in their relative abundance may lead to disease development. Detection methods like qPCR could only measure RNA levels at a single time point, but cannot detect dynamic changes in RNA levels. So this case is ideal for detection using our system.

如描述中所提到的，我们的系统具有活细胞原位RNA感测的优势。Chk1和Chk1-s的表达水平在细胞内呈现周期性变化，它们相对丰度的改变可能导致疾病的发展。像qPCR这样的检测方法只能测量单一时间点的RNA水平，而无法检测RNA水平的动态变化。因此，这个案例非常适合使用我们的系统进行检测。

For Sensor, this time mScarlet was chose as our basic gene and EGFP as our reporter gene, details are mentioned in the section of Optimization of our system. And as proposed in our basic design, the sequence between basic gene and reporter gene was designed by a program. For Chk1, complementary sensor sequence pairs with sequence 3 and for Chk1-s, Also, complementary sensor sequence pairs with the connection sequence of sequence 2 and 4.

对于Sensor，这次我们选择了mScarlet作为我们的基础基因，EGFP作为我们的报告基因，详细内容在"系统优化"部分提及。按照我们的基本设计，基础基因和报告基因之间的序列是由程序设计的。对于Chk1，互补的传感器序列与序列3配对；对于Chk1-s，互补的传感器序列则与序列2和4的连接序列配对。

Two different 2A peptide E2A and T2A were added to the Sensor to avoid unnecessary complications in primer design caused by identical sequences.

两种不同的2A肽段E2A和T2A被添加到Sensor中，以避免因相同序列而在引物设计中引起不必要的复杂性。

1 Booth, B. J.et al. RNA editing: Expanding the potential of RNA therapeutics.Molecular Therapy 31, 1533-1549 (2023).
2 Jiang, K.et al. Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR.Nature Biotechnology 41, 698-707 (2023).
3 Kaseniit, K. E.et al. Modular, programmable RNA sensing using ADAR editing in living cells.Nature biotechnology 41, 482-487 (2023).
4 Wang, X., Tian, X. & Marchisio, M. A. Logic circuits based on 2A peptide sequences in the yeast Saccharomyces cerevisiae.ACS Synthetic Biology 12, 224-237 (2022).
5 Sun, J.et al. Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae.Biotechnology and bioengineering 109, 2082-2092 (2012).
6 Albakri, M. B., Jiang, Y., Genereaux, J. & Lajoie, P. mScarlet for imaging in Saccharomyces cerevisiae. (2018).
7 Katrekar, D.et al. Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA editing activity and specificity.Elife 11, e75555 (2022).
8 Burnie, J. P., Carter, T. L., Hodgetts, S. J. & Matthews, R. C. Fungal heat-shock proteins in human disease.FEMS microbiology reviews 30, 53-88 (2006).
9 Carmelo, V. & Sá-Correia, I. HySP26 gene transcription is strongly induced during Saccharomyces cerevisiae growth at low pH.FEMS microbiology letters 149, 85-88 (1997).
10 Amorós, M. & Estruch, F. Hsf1p and Msn2/4p cooperate in the expression of Saccharomyces cerevisiae genes HSP26 and HSP104 in a gene‐and stress type‐dependent manner.Molecular microbiology 39, 1523-1532 (2001).
11 Chen, Q., Fang, Y., Zhao, H., Zhang, G. & Jin, Y. Transcriptional analysis of Saccharomyces cerevisiae during high-temperature fermentation.Annals of microbiology 63, 1433-1440 (2013).
12 UniProt: the Universal Protein Knowledgebase in 2023
13 Thon, V. J.et al. Coordinate regulation of glycogen metabolism in the yeast Saccharomyces cerevisiae. Induction of glycogen branching enzyme.Journal of biological Chemistry 267, 15224-15228 (1992).
14 Parrou, J. L.et al. Dynamic responses of reserve carbohydrate metabolism under carbon and nitrogen limitations in Saccharomyces cerevisiae.Yeast 15, 191-203 (1999).
15 Ding, J.et al. Pore-forming activity and structural autoinhibition of the gasdermin family.Nature 535, 111-116 (2016).
16 Valenti, M., Molina, M. & Cid, V. J. Human gasdermin D and MLKL disrupt mitochondria, endocytic traffic and TORC1 signalling in budding yeast.Open biology 13, 220366 (2023).
17 Schmitt, E., Paquet, C., Beauchemin, M., Dever-Bertrand, J. & Bertrand, R. Characterization of Bax-ς, a Cell Death-Inducing Isoform of Bax.Biochemical and biophysical research communications 270, 868-879 (2000).
18 Edlich, F.et al. Bcl-xL retrotranslocates Bax from the mitochondria into the cytosol.Cell 145, 104-116 (2011).
19 Priault, M., Camougrand, N., Kinnally, K. W., Vallette, F. M. & Manon, S. Yeast as a tool to study Bax/mitochondrial interactions in cell death.FEMS Yeast Research 4, 15-27 (2003).
20 Priault, M.et al. Investigation of the role of the C-terminus of Bax and of tc-Bid on Bax interaction with yeast mitochondria.Cell Death & Differentiation 10, 1068-1077 (2003).
21 Greenhalf, W., Stephan, C. & Chaudhuri, B. Role of mitochondria and C‐terminal membrane anchor of Bcl‐2 in Bax induced growth arrest and mortality in Saccharomyces cerevisiae.Febs Letters380, 169-175 (1996).
22 Clow, A., Greenhalf, W. & Chaudhuri, B. Under respiratory growth conditions, Bcl‐x (L) and Bcl‐2 are unable to overcome yeast cell death triggered by a mutant Bax protein lacking the membrane anchor.European journal of biochemistry 258, 19-28 (1998).
23 Manon, S., Chaudhuri, B. & Guérin, M. Release of cytochrome c and decrease of cytochrome c oxidase in Bax-expressing yeast cells, and prevention of these effects by coexpression of Bcl-xL.FEBS letters 415, 29-32 (1997).
24 Priault, M., Chaudhuri, B., Clow, A., Camougrand, N. & Manon, S. Investigation of bax‐induced release of cytochrome c from yeast mitochondria: Permeability of mitochondrial membranes, role of VDAC and ATP requirement.European Journal of Biochemistry 260, 684-691 (1999).
25 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.Proceedings of the National Academy of Sciences 109, 197-202 (2012). UniProt: the Universal Protein Knowledgebase in 2023

1 Booth, B. J.et al. RNA editing: Expanding the potential of RNA therapeutics.Molecular Therapy 31, 1533-1549 (2023).
2 Jiang, K.et al. Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR.Nature Biotechnology 41, 698-707 (2023).
3 Kaseniit, K. E.et al. Modular, programmable RNA sensing using ADAR editing in living cells.Nature biotechnology 41, 482-487 (2023).
4 Wang, X., Tian, X. & Marchisio, M. A. Logic circuits based on 2A peptide sequences in the yeast Saccharomyces cerevisiae.ACS Synthetic Biology 12, 224-237 (2022).
5 Sun, J.et al. Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae.Biotechnology and bioengineering 109, 2082-2092 (2012).
6 Albakri, M. B., Jiang, Y., Genereaux, J. & Lajoie, P. mScarlet for imaging in Saccharomyces cerevisiae. (2018).
7 Katrekar, D.et al. Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA editing activity and specificity.Elife 11, e75555 (2022).
8 Burnie, J. P., Carter, T. L., Hodgetts, S. J. & Matthews, R. C. Fungal heat-shock proteins in human disease.FEMS microbiology reviews 30, 53-88 (2006).
9 Carmelo, V. & Sá-Correia, I. HySP26 gene transcription is strongly induced during Saccharomyces cerevisiae growth at low pH.FEMS microbiology letters 149, 85-88 (1997).
10 Amorós, M. & Estruch, F. Hsf1p and Msn2/4p cooperate in the expression of Saccharomyces cerevisiae genes HSP26 and HSP104 in a gene‐and stress type‐dependent manner.Molecular microbiology 39, 1523-1532 (2001).
11 Chen, Q., Fang, Y., Zhao, H., Zhang, G. & Jin, Y. Transcriptional analysis of Saccharomyces cerevisiae during high-temperature fermentation.Annals of microbiology 63, 1433-1440 (2013).
12 UniProt: the Universal Protein Knowledgebase in 2023
13 Thon, V. J.et al. Coordinate regulation of glycogen metabolism in the yeast Saccharomyces cerevisiae. Induction of glycogen branching enzyme.Journal of biological Chemistry 267, 15224-15228 (1992).
14 Parrou, J. L.et al. Dynamic responses of reserve carbohydrate metabolism under carbon and nitrogen limitations in Saccharomyces cerevisiae.Yeast 15, 191-203 (1999).
15 Ding, J.et al. Pore-forming activity and structural autoinhibition of the gasdermin family.Nature 535, 111-116 (2016).
16 Valenti, M., Molina, M. & Cid, V. J. Human gasdermin D and MLKL disrupt mitochondria, endocytic traffic and TORC1 signalling in budding yeast.Open biology 13, 220366 (2023).
17 Schmitt, E., Paquet, C., Beauchemin, M., Dever-Bertrand, J. & Bertrand, R. Characterization of Bax-ς, a Cell Death-Inducing Isoform of Bax.Biochemical and biophysical research communications 270, 868-879 (2000).
18 Edlich, F.et al. Bcl-xL retrotranslocates Bax from the mitochondria into the cytosol.Cell 145, 104-116 (2011).
19 Priault, M., Camougrand, N., Kinnally, K. W., Vallette, F. M. & Manon, S. Yeast as a tool to study Bax/mitochondrial interactions in cell death.FEMS Yeast Research 4, 15-27 (2003).
20 Priault, M.et al. Investigation of the role of the C-terminus of Bax and of tc-Bid on Bax interaction with yeast mitochondria.Cell Death & Differentiation 10, 1068-1077 (2003).
21 Greenhalf, W., Stephan, C. & Chaudhuri, B. Role of mitochondria and C‐terminal membrane anchor of Bcl‐2 in Bax induced growth arrest and mortality in Saccharomyces cerevisiae.Febs Letters380, 169-175 (1996).
22 Clow, A., Greenhalf, W. & Chaudhuri, B. Under respiratory growth conditions, Bcl‐x (L) and Bcl‐2 are unable to overcome yeast cell death triggered by a mutant Bax protein lacking the membrane anchor.European journal of biochemistry 258, 19-28 (1998).
23 Manon, S., Chaudhuri, B. & Guérin, M. Release of cytochrome c and decrease of cytochrome c oxidase in Bax-expressing yeast cells, and prevention of these effects by coexpression of Bcl-xL.FEBS letters 415, 29-32 (1997).
24 Priault, M., Chaudhuri, B., Clow, A., Camougrand, N. & Manon, S. Investigation of bax‐induced release of cytochrome c from yeast mitochondria: Permeability of mitochondrial membranes, role of VDAC and ATP requirement.European Journal of Biochemistry 260, 684-691 (1999).
25 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.Proceedings of the National Academy of Sciences 109, 197-202 (2012). UniProt: the Universal Protein Knowledgebase in 2023