WetLab Results

中文

EN

1.Overview

2.Pre-Experiment

3.Editing System Optimization

4.Strain Security System

5.Dynamic monitoring of splice variant

Overview

After careful design of our system, we first conducted a pre-experiment for basic concept validation. Following the completion of the pre-experiment, the wet lab members were divided into three groups for subsequent experiments. The first group optimized the editing system in response to issues identified during the preliminary experiment, details can be found in the Editing System Optimization section. The second group was responsible for developing an Engineering Strain Security System based on RNAssay, aiming to use the RNAssay system to protect the company's trade secrets. The third group was tasked with developing a method for in vivo dynamic monitoring of splice variant using RNAssay.

Structure of our results

The End


Pre-Experiment

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.

Plasmid construction and transformation

Overview of plasmids

pADAR
Independent expression pCEV-ADAR1
pCEV-ADAR2
homologous recombination pUC19-ADAR1-C2
pUC19-ADAR2_MCP-C2
pUC19-C1

psensor(screened by Bleomycin) psensor(screened by URA)
pSensor1_Bleomycin pSensor1_URA
pSensor2_Bleomycin pSensor2_URA
pSensor3_Bleomycin pSensor3_URA
pSensor4_Bleomycin pSensor4_URA

Evidences of successful construction

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.

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.

1.pCEV-ADAR1

Step1. Design of plasmid

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.

Figure 1 Illustration of the pGEV-ADAR1

Step2. Expected fragment length and expected electropherogram:

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

Figure 2 Electrophoretogram simulation of pGEV-ADAR1

Step3. Electrophoretic result:

Figure 3 Electrophoretic result of pGEV-ADAR1

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.)

Step4. Sequencing results:

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.

Figure 4 Sequencing results of pCEV-ADAR1

2.pCEV-ADAR2

Step1. Design of plasmid

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.

Figure 5 Illustration of the pGEV-ADAR2

Step2. Expected fragment length and expected electropherogram:

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

Figure 6 Electrophoretogram simulation of pGEV-ADAR2

Step3. Electrophoretic result:

Figure 7 Electrophoretic result of pGEV-ADAR2

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

Step4. Sequencing results:

The sequencing results shown below were correct.

Figure 8 Sequencing results of pCEV-ADAR2-7

Figure 9 Sequencing results of pCEV-ADAR2-8

3. pUC19-C1

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

Figure 10 Illustration of the pUC19-C1

4.pUC19-ADAR1-C2

Step1. Design of plasmid

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.

Figure 11 Illustration of the pUC19-ADAR1-C2

Step2. Expected fragment length and expected electropherogram:

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

Figure 12 Electrophoretogram simulation of pUC19-ADAR1-C2

Step3. Electrophoretic result:

Figure 13 Electrophoretic result of pUC19-ADAR1-C2

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

Step4. Sequencing results:

The sequencing results shown below were correct.

Figure 14 Sequencing results of pUC19-ADAR1-C2

5.pUC19-ADAR2_MCP-C2

Step1. Design of plasmid

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.

Figure 15 Illustration of the pUC19-ADAR2_MCP-C2

Step2. Expected fragment length and expected electropherogram:

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

Figure 16 Electrophoretogram simulation of pUC19-ADAR2_MCP-C2

Step3. Electrophoretic result:

Figure 17 Electrophoretic result of pUC19-ADAR2_MCP-C2

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

Step4. Sequencing results:

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

Figure 18 Sequencing results of pUC19-ADAR2_MCP-C2-1

Figure 19 Sequencing results of pUC19-ADAR2_MCP-C2-2

6~9.pSensor1~4(Bleomycin)

Figure 20 Illustration of 4 pSensors screened by bleomycin

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).

10. pSensor1_URA

Step1. Design of plasmid

Figure 21 Illustration of pSensor1_URA_Pre

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.

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.

Step2. Sequencing results:

Figure 22 Sequencing results of pSensor1_URA_Pre

11. pSensor2_URA

Step1. Design of plasmid

Figure 23 Illustration of pSensor2_URA_Pre

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.

Step2. Sequencing results:

Figure 24 Sequencing results of pSensor2_URA_Pre-1

Figure 25 Sequencing results of pSensor2_URA_Pre-2

12. pSensor3_URA

Step1. Design of plasmid

Figure 26 Illustration of pSensor3_URA_Pre

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.

Step2. Sequencing results:

Figure 27 Sequencing results of pSensor3_URA_Pre-1

Figure 28 Sequencing results of pSensor3_URA_Pre-2

13. pSensor4_URA

Step1. Design of plasmid

Figure 29 Illustration of pSensor4_URA_Pre

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.

Step2. Sequencing results:

Figure 30 Sequencing results of pSensor4_URA_Pre-1

Figure 31 Sequencing results of pSensor4_URA_Pre-2

Successful yeast transformation

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.

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

Figure 32 Electrophoretic result of yeast in Pre-experiment

Naming rules: H stands for homologous recombination; I stands for independent expression; A1-P1 stands for yeast introduced with ADAR1_p150 and pSensor1, A2-P1 stands for yeast introduced with ADAR2_MCP and pSensor1, and from right to left they are 1-8 (1-6 for independent expression)

Characterization results

LSCM (laser scanning confocal microscope) Analysis

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

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.

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.

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.

Figure 33 Fluorescence of ADAR1_p150

Figure 34 Fluorescence of ADAR2_MCP

Flow cytometry analysis

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.

Figure 35 Gate Settings for Flow Cytometry Analysis

Figure 36 The result of Flow cytometry analysis

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.

qPCR

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.

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.

Figure 37 The qPCR rusult of ADAR1_p150

Figure 38 The qPCR rusult of ADAR2_MCP

Western Blot

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.

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.

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

First experiment

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.

yeast ADAR Form of expression promoter
Blank \ \ \
A1_H_P ADAR1 homologous recombination PDC1
A2_H_P ADAR2 homologous recombination PDC1
A1_I_H ADAR1 Independent expression HXT7

Standard protein concentration solutions were prepared, and the OD562 values of the standard samples were measured. A linear regression was performed to fit a function relating protein concentration to OD562.

Standard protein concentration(mg/mL) OD562
0 0.811
0.025 0.933
0.05 1.018
0.1 1.192
0.2 1.438
0.3 2.141
0.4 2.559
0.5 2.94

Figure 39-1 Standard protein sample concentration absorbance

The OD562 values of the samples obtained from cell lysis were measured. Using the linear function, the protein concentration was approximated, and the volume of each sample to be added was calculated to ensure that the total protein amount loaded in each electrophoresis lane was approximately equal.

Sample concentration(mg/mL) Blank A1_H_P A2_H_P A1_I_H
total 0.38166 0.41249 0.55968 0.56893
supernatant 0.42790 0.48955 0.57124 0.63367
precipitate 0.02568 0.02938 0.02953 0.03354

Add sample volume(μL) Blank A1_H_P A2_H_P A1_I_H
total 16.00 14.80 10.91 10.73
supernatant 14.27 12.47 10.69 9.64
precipitate 16.00 14.80 10.91 10.73

After electrophoresis, transfer to a solid membrane, antibody binding, washing, and fluorescent detection, the resulting protein fluorescence images were as follows:

Figure 39-2 Western blot results of the first experiment

Naming rule: '- T' represents total, '- S' represents supernatant, and' - P 'represents precipitate

Second experiment

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.

yeast ADAR Form of expression promoter
Blank \ \ \
A1_H_P ADAR1 homologous recombination PDC1
A2_H_P ADAR2 homologous recombination PDC1
A1_H_T ADAR1 homologous recombination TEF
A2_H_T ADAR2 homologous recombination TEF

The experimental procedure was the same as described above, and the results are as follows:

Figure 39-3 Western blot results of the second experiment

Naming rules: '- T' represents total, and '- S' represents supernatant

Conclusion and Discussion

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.

Subcellular Localization of ADAR1 and ADAR_MCP

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.

Plasmid construction and transformation

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.

Figure 40 Illustration of the pLocate-ADAR1

Figure 41 Illustration of the pLocate-ADAR_MCP

Plasmid construction

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:

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.

Electrophioult:

Figure 42 Electrophoretic result of pLocate-ADAR1

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.

Figure 43 Electrophoretic result of pLocate-ADAR_MCP

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.

Figure 44 Sequencing results of pLocate-ADAR1

Figure 45 Sequencing results of pLocate-ADAR_MCP

The sequencing results show that the plasmid construction is correct.

Yeast Transformation

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.

Figure 46 Illustration of homologous recombination

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.

Figure 47 Electrophoretic result of pLocate-ADAR1

Figure 48 Electrophoretic result of pLocate-ADAR_MCP

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.

Fluorescence Observation Using LSCM

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.

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.

Fluorescence microscopy results are presented below:

Figure 49 Fluorescence of yeast introducing pLocate-ADAR1

Figure 50 Fluorescence of yeast introducing pLocate-ADAR_MCP

ADAR toxicity verification

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.

Experimental Design

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.

Yeast ADAR Promoter Expression Intensity
B \ \ \
A1 ADAR1_p150 PDC1 Low
A2 ADAR2_MCP PDC1 Low
T1 ADAR1_p150 TEF High
T2 ADAR2_MCP TEF High

Experimental Results

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

Figure 51 Sample 1, initial concentration OD600=0.1

Figure 52 Sample 2, initial concentration OD600=0.01

Figure 53 Sample 3, initial concentration OD600=0.001

Figure 54 Sample 4, initial concentration OD600=0.0001

Note: The third and fourth groups did not show significant colony formation during the first 19 hours due to their low initial concentrations.

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.

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.

Discussion and conclusion for ADAR toxicity verification

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.

Figure 55 Preliminary experiment results (OD600=0.001, 26h)

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:

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.

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.

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.

Discussion and Conclusion

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.

The End


Editing System Optimization

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.

Promoter replacement

Successfully constructed plasmids and transformed into yeast system

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

plasmid construction

Step1. Design of plasmid :

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.

Figure 56 Illustration of TEF-C1

Figure 57 Illustration of TEF-C2-ADAR1

Figure 58 Illustration of TEF-C2-ADAR_MCP

Step2. Sequencing results:

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

Figure 59 Sequencing results of TEF-C1, TEF-C2-ADAR1 and TEF-C2-ADAR_MCP

Detection of transcription levels

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.

Figure 60 The comparison of transcription level between TEF1-ADAR and PDC1-ADAR

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.

Adding MS2

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.

Figure 61 table of plastids in 'Adding MS2' Part

Successfully constructed plasmids and transformed into yeast system

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

plasmid construction

Step1. Design of plasmid :

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

Figure 62 Illustration of pSensor-MS2-0, pSensor-MS2-2, pSensor-MS2-4

Step2. Sequencing results:

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

Figure 63 Sequencing results of pSensor-MS2-0, pSensor-MS2-2, pSensor-MS2-4

Successful yeast transformation

Yeast transformation was successful

Test results

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

Figure 64 FACs Results of Editing Systems Containing Different MS2 sequences

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.

Figure 65 Confocal Results of Editing Systems Containing Different MS2 sequences

Figure 66 FACs Results of Editing Systems Containing Different ADAR Types and Promoters

Figure 67 Confocal Results of Editing Systems Containing Different ADAR Types and Promoters

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

Quantitative characterization of editing intensity

Successfully constructed plasmids and transformed into yeast system

plasmid construction

Step1. Design of plasmid :

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.

Figure 68 Illustration of Xyl-pSensor-HO-C1, Xyl-pSensor-HO-C2

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

Figure 69 Illustration of homologous recombination

Step2. Sequencing results:

Figure 70 Sequencing results of Xyl-pSensor-HO-C1, Xyl-pSensor-HO-C2

Results

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.

Figure 71 The Relationship Between Relative Transcription Level and Editing Efficiency

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.

The End


Strain Security System

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.

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.

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.

Figure 72 Illustration of plastids in Strain Security System

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.

Figure 72-1 Electrophoretic results of pSensor-URA-Cre and others

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

Figure 72-2 Sequencing results of pSensor-URA-Cre

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.

The End


Application for in vivo dynamic monitoring of splice variant

Successfully constructed plasmids and transformed into yeast system

Overview of plasmids

(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

Evidences of successful construction of plasmids

1. pSensor-target_Chk1-Chk1

Step1. Design of plasmid:

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.

Figure 73 Illustration of the pSensor-target_Chk1-Chk1

Step2. Electrophoretic results:

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.

The correct band will be 1000bp long.

Figure 74 Electrophoretic result of pSensor-target_Chk1-Chk1

Step3. Sequencing results:

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

Figure 75 Sequencing results of pSensor-target_Chk1-Chk1

2. pSensor-target_Chk1-Chk1s

Step1. Design of plasmid:

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.

Figure 76 Illustration of the pSensor-target_Chk1-Chk1s

Step2. Electrophoretic results:

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

Figure 77 Electrophoretic result of pSensor-target_Chk1-Chk1s

Step3. Sequencing results:

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

Figure 75 Sequencing results of pSensor-target_Chk1-Chk1s

3. pSensor-target_Chk1s-Chk1

Step1. Design of plasmid:

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..

Figure 79 Illustration of the pSensor-target_Chk1s-Chk1

Step2. Electrophoretic results:

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.

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.

Figure 80 Electrophoretic result of pSensor-target_Chk1s-Chk1

Step3. Sequencing results:

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

Figure 81 Electrophoretic result of pSensor-target_Chk1s-Chk1

4. pSensor-target_Chk1s-Chk1s

Step1. Design of plasmid:

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.

Figure 82 Illustration of the pSensor-target_Chk1s-Chk1s

Step2. Electrophoretic results:

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.

Figure 83 Electrophoretic result of pSensor-target_Chk1s-Chk1s

Step3. Sequencing results:

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

Figure 84 Sequencing results of pSensor-target_Chk1s-Chk1s

Successful yeast transformation

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.

Figure 85~88 Electrophoretic result of yeast

Naming rules: A1=ADAR_p150, A2=ADAR2_MCP,KK=pSensor-target_Chk1-Chk1, SS=pSensor-target_Chk1s-Chk1s, KS=pSensor-target_Chk1-Chk1s, SK=pSensor-target_Chk1s-Chk1, TEF is the new promoter we use, and the groups with ms0 or ms2 are the results for another part of our project, we did the yeast transformation together.

Characterization results

LSCM(laser scanning confocal microscope)

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.

Figure 89 Fluorescence of yeast introducing Sensor-target_Chk1(s)-Chk1(s)(ADAR1)

Figure 90 Fluorescence of yeast introducing Sensor-target_Chk1(s)-Chk1(s)(ADAR2)

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.

Flow cytometry analysis

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.

Figure 91 Gate Settings for Flow Cytometry Analysis

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.

Figure 92

Figure 93

Figure 94

Then we processed the datas into bar chart following the formula: Edit Efficiency = 100×mScarlet&eGFPmScarlet%

Figure 95 FACs Results of Sensor-target_Chk1(s)-Chk1(s) (ADAR1)

Figure 96 FACs Results of Sensor-target_Chk1(s)-Chk1(s) (ADAR2)

According to the datas, we came to some conclusions:

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).

Figure 97 IntaRNA Simulation of Binding Energy of Chk1(s)-Chk1(s)

Naming rules: TA1=TEF-ADAR1, TA2=TEF-ADAR2

Discussion

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

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.

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

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 )

Conclusion

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.

Reference

[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.

The End