Team:SIAT-SCIE/Results

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We provide models aiming to provide better understanding of our project and result analysis

1. The Structure and Domain of CjCas9

To provide a better view of the structure and functional domain of CjCas9, we provide a 360-degree rotating graph of the protein with its sgRNA and target DNA.

Figure.1 Structural image of CjCas9 in complex with sgRNA(red) and target DNA(purple) sequence (with PAM AGAAACC). We have annotated the protein by color according to their specific functions[1]. The white REC1 domain and the grey REC2 domain form the REC (crRNA recognition) domain. The cyan RuvC domain, the blue PLL domain, the yellow WED domain and the incarnadine PI domain form the NUC(nuclease) domain. Further explanation about the functions of these region can be found at Yomada 2017.

The display is created using the free online software Jmol[1] to vistualize the 3D structure of CjCas9, with the help of PicGIF Lite to transform images into gif.

2. Estimated Number of Cas9 Encapsulation by OMVs

OMVs have diameters that vary from 20 to 250 nanometers[3], which allows it to carry great amount of substance. To provide a more statistical explanation of the Cas9 carrying ability of OMVs, we use a simulant model to calculate the amount of Cas9 proteins one OMV can carry. The approximate volume of CjCas9, approximately 700 nanometers cube(82x96x88), is determined by the bounding box volume obtained by Jmol.

In addition, as supplementary material for our choice in the type of Cas9s, we also estimated the number of SgCas9s[4] one OMV can cargo through the same method. The result shows an estimated bounding box volume of 1430 nanometers cube. The difference between the results is shown in Figure.2. As shown, CjCas9’s small size allows it to be carried in great amount during the transmission and is, therefore, more suitable to be used in OMV-mediated gene editing.

Figure.2 Estimated quantity of Cas9s encapsulation by OMVs. The data is calculated assuming cuboid shape of Cas9 proteins and perfect sphere shape of OMVs.

FURTHER Results

1. Testing torA protein

The signal peptide torA increases the amount of cjCas9 protein in the cytoplasm that can be transferred to the periplasm, thereby increasing the likelihood that cjCas9 is wrapped by OMV.
In order to test the expression and function of torA, GFP-mut3 was used instead of cjCas9 since we can directly observe the effect of torA by gauging the fluorescence level in cytoplasm and periplasm respectively to determine the amount of GFP-mut3 protein successfully transferred to periplasm, which is indicative of the efficiency of torA. The coding sequence of torA signal peptide precedes the coding sequence of cjCas9.
We performed Osmoshock in order to separate the cytoplasm and periplasm of GFP-mut3- sequence-carrying bacteria. The result of the fluorescence test in Figure 1.1 and 1.2 shows that the torA signal peptide significantly increases the efficiency of the transportation of GFP-mut3 into the periplasm in both strains of bacteria.
This indicates that torA signal peptide is capable of helping transport proteins such as cjCas-9 into the periplasm, which is the step that just precedes cjCas-9’s encapsulation by OMVs.

2. Increased production of OMV by using ompA-knockout bacteria

In order to increase the amount of OMV produced in the periplasm to optimize our design, the outer membrane protein A precursor (ompA) is knocked out to induce hyper vesiculation. The result from Figure 2 indicates that in both experiments, the ΔPF/TF value of BW25113, which is a common strain of E.coli, is larger than that of ompA-knockout BW25113. This reflects that the ompA-knockout bacteria produced more OMVs, which may wrap the GFP-mut3 protein in the periplasm, thereby causing a decrease in a measurement of fluorescent value in the periplasm.

To further prove that the GFP can be successfully wrapped by OMV, we isolate the OMV in periplasm after osmoshock and measure the fluorescent value in it. The positive result in the graph reflects that our hypothesis is correct, and indicates that OmpA knock-out bacteria will produce more OMVs than normal bacteria since the fluorescent value is higher in it.

Reference [1]: Yamada, M., Watanabe, Y., Gootenberg, J. S., Hirano, H., Ran, F. A., Nakane, T., . . . Nureki, O. (2017). Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems. Molecular Cell, 65(6).
[2]: Jmol: an open-source Java viewer for chemical structures in 3D. http://www.jmol.org/
[3]: Schwechheimer, C., & Kuehn, M. J. (2015). Outer-membrane vesicles from Gram-negative bacteria: Biogenesis and functions. Nature Reviews Microbiology, 13(10), 605-619.
[4]: Huai, C., Li, G., Yao, R., Zhang, Y., Cao, M., Kong, L., . . . Huang, Q. (2017). Structural insights into DNA cleavage activation of CRISPR-Cas9 system. Nature Communications, 8(1).