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Due to its high specificity, safety, and sustainability, we have chosen RNAi technology for the treatment of tomato gray mold disease. After screening, we selected BcchsIIIa, Bccyp51, BcOAH1, and Bcpme1 as our targets for RNAi. Since shRNA is known to have higher stability in natural environments and can be produced on a large scale at low cost using fermentation engineering in the laboratory, we have selected the most common strain, E. coli, for fermentation production. We used E. coli HT115(DE3), which lacks nucleases. The designed target sequences are constructed through the order of sense fragment loop-antisense fragment, and connected to the plasmid pET-28a(+) to obtain our shRNA.

To verify our successful transformation, we used specific primers to amplify the plasmids extracted from E. coli by PCR, and demonstrated that our shRNA expression vector successfully entered E. coli HT115(DE3) by agarose gel electrophoresis (Figure1). Subsequently, RNA was extracted from E. coli after IPTG induction, and the successful production of shRNA in E. coli was confirmed by the size of the bands.

In our experiments, we used shRNA(gfp) as the control group.The produced shRNA was sprayed on the tomato fruits infected with B. cinerea, and we explored the inhibitory effect of shRNA on gray mold infection in tomatoes by measuring the distribution of disease spots (Figure 2a). We also detected the inhibitory effect of shRNA on target genes using qRT-PCR (Figure 2b). The results indicate that on the third day, the highest silencing rate of the target gene achieved by spraying shRNA(Pme1)-2 was 59%.

We found that the inhibitory effect of shRNA on gray mold disease was not as good as we had expected in actual use, which may be because shRNA is still not very stable in natural environments and the probability of cell uptake and processing of shRNA is not high. Faced with this problem, we must find a more effective system to deliver our shRNA.

Cell-penetrating peptides (CPPs) have been shown to penetrate the plasma membrane of various plant species and tissues. Multiple studies have demonstrated that CPPs can enhance the stability of shRNA and significantly improve the efficiency of silencing through CPP-mediated sRNA delivery systems. Ultimately, we have chosen BP100-(KH)₉ as our CPP and allowed it to bind to shRNA, hoping that it will confer greater stability to our shRNA under natural conditions and enhance the efficiency of gene silencing on the target gene.

To obtain the BP100-(KH)₉+shRNA complex, we incubated CPP and shRNA in a fixed ratio at room temperature. For the binding of BP100-(KH)₉ with shRNA, we observed naked shRNA (Figure 3a), naked BP100-(KH)₉ (Figure 3b), and BP100-(KH)₉+shRNA complex (Figure 3c)under scanning electron microscopy (SEM), and significant morphological differences were observed among the three. The presence of spherical aggregates in Figure 3c suggests the successful binding of BP100-(KH)₉ with shRNA.
To learn more detailed information about shRNA, BP100-(KH)₉, and shRNA+CPP under SEM, please click on Proof of Concept

We sprayed shRNA complexed with BP100-(KH)₉ onto tomato fruits infected with B. cinerea and explored the inhibitory effect of BP100-(KH)₉+shRNA on gray mold disease by measuring the distribution of disease spots (Figure 4a). We also detected the inhibitory effect of BP100-(KH)₉+shRNA on the target gene using qRT-PCR (Figure 4b). From the results, it can be observed that BP100-(KH)₉+shRNA significantly enhanced the efficiency of shRNA-mediated gene silencing on the target gene. Among them, BP100-(KH)₉ showed the highest increase in silencing rate against Bccyp51. The silencing rate of Bccyp51 increased from 51% to 66%.

Furthermore, through continuous qRT-PCR analysis, we found that the highest silencing rate of the target gene by BP100-(KH)₉+shRNA was achieved at the 60th hour (Figure 5).

For more information on continuous qRT-PCR, please refer to Proof of Concept.

Additionally, the time-dependent efficiency of BP100-(KH)₉+shRNA-mediated gene silencing observed in the experiment is consistent with our RNAi model.
For more information on the model, please see the Model

Although it has been demonstrated that BP100-(KH)₉+shRNA-mediated gene silencing of individual target genes has shown promising efficacy in the treatment of tomato gray mold disease, we are still not entirely satisfied with the inhibitory effect of a single shRNA on the infection of B. cinerea, especially the unsatisfactory silencing effect on the BcchsIIIa gene. We believe that this situation may be attributed to instability of a single shRNA, among other factors, ultimately limiting the optimal effectiveness of single-target gene silencing by shRNA.
Moreover, in order to achieve better suppression of gray mold, a large amount of single shRNA is required, which also reminds us to always consider the cost issue of shRNA.

We have learned that the bi-shRNA design leads to more rapid onset of gene silencing, higher efficacy, and greater durability when compared with either siRNA or conventional shRNA. By concatenating shRNA molecules targeting different mRNA sequences, the resulting shRNAs can be processed into different siRNAs that act simultaneously. Moreover, compared to mixing and spraying different shRNAs, by concatenating different shRNAs to obtain bi-shRNA, we can save more on the usage of shRNAs while achieving better silencing effects. As shown in the Figure 6, we used website prediction again to optimize shRNAs targeting BcchsIIIa and Bccyp51, resulting in shRNA(CHSIIIa)* and shRNA(cyp51)*, respectively. We then concatenated them to construct bi-shRNA named Box-survival, which collectively silences two important genes essential for the survival of B. cinerea. During application, we will also combine it with cell-penetrating peptide BP100-(KH)₉ to construct BP100-(KH)₉+bi-shRNA for optimal inhibitory effects.

To validate the successful production of Box-survival in E. coli, we constructed a plasmid and transformed it into E. coli for large-scale cultivation. After induction with IPTG, we extracted RNA from E. coli and confirmed the successful generation of shRNA based on the band size observed on the gel. In the gel image, there is a bright band between lanes 100 and 150, which corresponds to the size of 124bp. This is because Box-survival is a concatenation of two shRNAs, resulting in a larger size compared to the designed single-target shRNA (69bp), showing a significant molecular weight difference.

Similarly, we sprayed BP100-(KH)₉ + bi-shRNA on tomato fruits infected with B. cinerea and compared the inhibitory effects of BP100-(KH)₉ + bi-shRNA with our selected four single-target BP100-(KH)₉ + bi-shRNA by measuring the distribution of disease spots (Figure 8a). We also examined the inhibitory effect of BP100-(KH)₉ + bi-shRNA on the target genes of B. cinerea using qRT-PCR, comparing it with BP100-(KH)₉ + shRNA (Figure 8b). From the results, it can be observed that the optimized sequences of shRNA(CHSIIIa)* and shRNA(cyp51)* exhibit lower off-target probabilities compared to the original sequences. Furthermore, compared to BP100-(KH)₉ + shRNA, BP100-(KH)₉ + bi-shRNA demonstrate a stronger inhibitory effect on gray mold disease. From the Figure 8b, we can see that among all shRNAs, bi-shRNA(Box-survival) showed the highest silencing efficiency against the target gene, reaching 70%.

According to the results mentioned above, our BP100-(KH)₉ + bi-shRNA has shown excellent inhibitory effects on the target gene with lower off-target effects. Therefore, we can ensure that BP100-(KH)₉ + bi-shRNA can be used as our RNAi product for field spraying, achieving precise, efficient, and powerful attack on pathogenic fungi. The engineering cycle of our RNAi silencing system also concludes after this validation step.

In order to address the instability of RNAi biopesticides and regulate the amount of pesticide used, we hope to package our product in a specific form to avoid misuse. We drew inspiration from the design of laundry pods and learned that they are made up of individual beads wrapped in PVA film. Through investigation, we found that PVA film has good water solubility, biocompatibility, degradability, high mechanical strength, ease of processing, low production cost, and easy availability. It also has barrier and anti-static properties, which can effectively prevent RNA from coming into contact with nucleases in the environment and being degraded. Therefore, we decided to use PVA film to wrap our CPP-shRNA formulations and the engineered bacterial host B. subtilis.

As the produced CPP-shRNA complexes exist in the form of an aqueous solution, we need to overcome the water solubility of PVA. To achieve this, we plan to use a powder formulation with strong water-absorbing capability, which does not affect the stability of the CPP-RNA complex, to encapsulate CPP-shRNAs within the beads. Our initial consideration is agarose, a commonly used inert material for nucleic acid electrophoresis in laboratories. Agarose is able to absorb water without introducing large charged groups, thus maintaining the stability of nucleic acids and meeting our basic requirements. Our engineered bacteria are in the form of bacterial powder, and users are required to add sucrose for reconstitution.

In our design of Polycobead 1.0, we mix the CPP-shRNA aqueous solution with agarose commonly used in laboratories, and then combine it with the engineered bacterial powder before packaging them in water-soluble PVA film. The resulting pods enhance the stability of the CPP-shRNA complexes and can dissolve rapidly in water for direct application.

We drop a certain concentration of CPP-shRNA aqueous solution onto agarose powder, and then mix the engineered bacterial powder of B. subtilius with the agarose powder. Finally, we package these mixed powders into a certain size of PVA film, and seal them using a heat sealing machine, resulting in our Polycobead 1.0 (Figure 9).

To understand the structure of our CPP-shRNAs in aqueous solution, we observed the bare shRNAs, bare CPPs and CPP-shRNAs by scanning electron microscopy (SEM). From the scanning electron microscope (SEM) results, it can be seen that the naked shRNAs samples showed white spots of different sizes under the scanning electron microscope, which may be caused by the aggregation of shRNAs and the concentration of aggregation is different. And at higher magnification, the white spots will disappear more as the magnification increases, probably because the electron energy of the electron beam is too strong.

Samples of bare CPPs were examined under a scanning electron microscope and appeared as dotted white spots in the field of view. We speculate that these white spots are aggregates of our cell-penetrating peptides.

Finally, our CPP-shRNAs samples were observed by scanning electron microscopy (SEM). Many spherical polymers of different sizes were observed in the experiment, and smaller clumps of spherical polymers were observed after magnifying individual polymers.

At the same time, in order to test that our PVA film can dissolve smoothly in water and release our RNAi pesticide, we put our prepared coagulation beads into water, and the results proved that the beads dissolved in water after about 1min. The solution in the beads is released.

It is necessary to improve the release efficiency of bio-pesticides and simplify the use of pesticides. From the design and testing results of our PolycoBead1.0, we identified the following limitations:

• 1. As a water-absorbing material, agarose cannot be completely dissolved in water at room temperature. The water insolubility of agarose leads to the formation of colloid that can cross-link CPP-shRNAs, resulting in the deposition of agarose at the bottom or the formation of insoluble precipitates after spraying on farmland, resulting in the inability to completely release the active ingredients of pesticides.


• 2. Because we added a suicide switch in the engineered bacteria and the strain needed to be resuscitating, the farmers needed to add additional sucrose to survive and function, which caused a certain amount of inconvenience.


• 3. The price of agarose is expensive. If it is used as an auxiliary material in the coagulation beads, the cost of a single bead will be increased, which makes it difficult to promote in the market.

In order to solve the above problems, we decided to iterate on the auxiliary components of coagulation beads to give users a more effective, convenient and affordable experience.

The engineering bacteria, sucrose and 1% sodium alginate solution were mixed and dropped into Ca²⁺ solution. The sodium alginate reacted with calcium ions to form a water-insoluble calcium alginate shell in the outer layer, while the engineering bacteria and sucrose were wrapped in calcium alginate to form smaller beads. These beads were added to glycerol containing CPP-shRNAs, encapsulated in PVA films of certain size, and finally sealed using a heat sealer to obtain our Polycobead 2.0.

The production process of PolycoBead 2.0.

To ensure that the activity of CPP-shRNA could be maintained in the glycerol system, we used the glycerol system containing CPP-shRNA to verify by agarose gel electrophoresis. As shown in the figure, the lanes from left to right are Marker, naked shRNA aqueous solution, naked shRNA glycerol-aqueous solution (glycerol: water = 9:1), CPP-shRNA complex aqueous solution, CPP-shRNA complex glycerol-aqueous solution (glycerol: water = 9:1), and Marker repectively. It can be seen from the second and third lanes that glycerol has a certain blocking effect on naked shRNA, but it does not completely affect its movement in electrophoresis. Showing in the fourth and fifth lanes, after combining with CPP, shRNA would form a certain complex structure with CPP and could not move in electrophoresis, while mixing with glycerol did not affect the binding between the two. Meanwhile, we also verified by SEM that the structure of CPP-shRNA in glycerol system does not change.

To demonstrate that when the calcium alginate-engineered bacteria complex is released into the environment, the engineered bacteria in the complex will release into the soil at a slow rate along with the loss of Ca²⁺, and we have verified that the calcium alginate-engineered bacteria could release the liquid in the soil within 40h. We hope that PolycoBead2.0 can be applied by simply stirring it in water, and the effective constituent will be released to work. The release time of PolycoBead 2.0 was tested and it could be released in 5L water within 2min.

Click Proof of concept for more details.

Finally, we also tested that the effective constituent in PolycoBead 2.0 could play a good role, so we sprayed the solution with PolycoBead 2.0 on tomatoes infected with B. cinerea, and it could be obviously observed that the solution could play a good inhibitory effect on B. cinerea.

It can be concluded from the above results that our PolycoBead can stably encase CPP-shRNA and engineered B. subtilis, then release it stably after dissolving in water, and play a good role in tomato infected with B. cinerea. Therefore, we can prove that PolycoBead 2.0 as our final product can maintain the activity of the effective constituent during storage, show good results in application, and bring more benefits and convenience to farmers when used.