Difference between revisions of "Team:TJU China/Notebook/Group1"

After a lot of document viewing and brainstorms, we finally came up with a clear idea about how we were going to carry out our part of project. We decided to construct the RNPs (complex of sgRNA and Cas9 protein) and to wrap them with delivery vector in order to improve the gene-editing efficiency of the system into the cells.

After daily meetings and discussions, we exchanged knowledge and ideas from the literatures we read. Comparing different transportation methods, including physical electroporation, chemical nanoparticles, and even virus methods, we decided to improve the transport efficiency of the system by using two-dimensional nanomaterials to encapsulate the cas9/sgRNA complex assembled in vitro to transport into the cells.

We began to delve into more detailed and specific knowledge, such as the structure of cas9 protein and how it works, the structure and composition of sgRNA and how it is assembled with cas9 protein, the method of primer design, learning how to design sgRNA, and so on.

After confirming the type of cas9 protein, we started buying and asking other labs if they had the same cas9 protein. While waiting for an answer, we are continuing to refine the questions left over from last week's knowledge and begin to learn the experimental procedures and necessary knowledge related to protein expression purification.

We continued to search for related documents in order to choose the appropriate target sequences of gene for our project. After the final decision of making eGFP as our interested sequence of gene, we immediately sent out the primers of sgRNA we designed to GENEWIZ, and we also sent lots of emails to different labs to see if they can offer us a stable cell strain expressing eGFP protein.

Meanwhile, we successfully transformed the plasmid pET-NLS-Cas9-6xHis (purchased from Addgene) into E.coli (BL21), then we tried to express and purify the cas9 protein according to the protocol[1]. But the results were not so ideal.
【1】Ha J S, Lee J S, Jeong J, et al. Poly-sgRNA/siRNA ribonucleoprotein nanoparticles for targeted gene disruption[J]. Journal of Controlled Release, 2017, 250:27-35.

PCR and Gel Purification (TIANgel Midi Purification Kit) of the template of sgRNA, we tried different Tm temperatures.

After two weeks of hard working, we got our first batch of Cas9 protein.
At the same time, we tried our first transcription and preparation of sgRNA using T7 High Efficiency Transcription Kit and EasyPure RNA Purification Kit (TransGene Biotech) but failed. We supposed that the template might be polluted by RNase during the process.

We searched for information and knowledge of experiments dealing with RNA and then retried the PCR and Gel Purification of the template according strictly to the RNA principles. However, we failed again.
On the other hand, we managed to get plasmid encoding AcGFP by using TIANprep Mini Plasmid Kit (TIANGEN).

We confirmed the best Tm temperature of PCR was 67.2℃ after several preliminary experiments. And this time, there were lines shown on the RNA gel, but they were not clear. And the RNA marker (TAKARA) didn’t appear exactly as the instruction. Then we moved forward to the In vitro digestion of DNA but failed. Since that the result of the SDS-PAGE of Cas9 protein showed no problem, we considered that the sgRNA might degraded.

Line1，plasmid；2，cleavage result

test10.4