Team:TJU China/Notebook/Group3

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Mar.

Apr.

May

Jun.

Jul.

Aug.

Sep.

Oct.

March

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.

April

week3

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.

week4

Meanwhile, we successfully transformed the plasmid pET-NLS-Cas9-6xHis into E.coli (Rosetta), then we tried to express and purify the cas9 protein according to the protocol. But the results were not so ideal, Cas9 protein degraded.

Figure 1.Plate of Rosetta transfected with pET-NLS-Cas9-6xHis plasmid.

May

week1

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

Figure 2.Assembly of template of sgRNA using PCR. Lane M, marker. Lane 1, Tm=65.2℃. Lane 2, Tm=66.2℃. Lane 3, Tm=66.8℃. Lane 4, Tm=67.2℃. Lane 5, Tm=68.2℃.

week2

After two weeks of hard working, we got our first batch of Cas9 protein.

Figure 3.Result of purification by Affinity chromatography (Ni-NTA) . Lane M, marker. Lane 1, before eluted by Buffer A. Lane 2, after eluted by Buffer A. Lane 3, before eluted by Buffer B. Lane 4, after eluted by Buffer B. Buffer A(50 mM Tris-HCl (pH 8.0), 1 M NaCl, 20% glycerol, 2 mM TCEP and 20 mM imidazole). Buffer B( 50 mM Tris-HCl (pH 8), 1 M NaCl, 20% glycerol, 2 mM TCEP and 500 mM imidazole).

Figure 4.SDS-PAGE result of ion exchange. Lane M, marker. Lane 1, Cas9 protein after ion exchange purification.

Figure 5.SDS-PAGE result of gel filtration. Lane M, marker. Lane 1, Cas9 protein after gel filtration purification.

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.

week3

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 eGFP by using TIANprep Mini Plasmid Kit (TIANGEN).

Figure 6.Extraction of eGFP plasmid. Lane M, marker. Lane 1, eGFP plasmid.

week4

We confirmed the best Tm temperature of PCR was 67.2℃ after several preliminary experiments. And this time, there were Lanes 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.

Figure 7.In vitro cleavage of plasmid. Lane M, marker. Lane 1, eGFP plasmid. Lane 2, sgRNA:Cas9:DNA=10:10:1.

June

Preparation for the final-term exams.

July

week1,2,3

Most members joined a summer camp as a part of school lessons.

week4

In order to reduce the influence of RNase on the subsequent experiment, the phenol-chloroform method was used to remove RNase. And after several experiments, we confirmed that the best reaction time for transcription was 16 hours.

August

week1

We tried different ways of purification of the transcription products——EasyPure RNA Purification Kit and phenol-chloroform purification. And thanks to our continuous efforts, we managed to run the nucleic acid gel electrophoresis of sgRNA perfectly.

Figure 8. In vitro transcription of sgRNA. Lane M, marker. Lane 1, 100ng sgRNA. Lane 2, 200ng sgRNA.

week2

We succeeded in the In vitro digestion of DNA but the efficiency was not as good as we expected, so we began to find the reason and solutions to improve the cleavage efficiency. The experiments was carried out according to the protocol (NEB, In vitro digestion of DNA with EnGen Cas9 NLS, S. pyogenes).

Figure 9.The effect of different components on the cleavage reaction. Lane M, marker. Lane 1, eGFP plasmid. Lane 2, plasmid+sgRNA. Lane 3, plsmid+Cas9 protein. Lane 4, plasmid+reaction buffer.

week3

We got plasmid of better quality.

Figure 10.Extraction of eGFP plasmid. Lane M, marker. Lane 1, eGFP plasmid. We also designed the experiments in order to improve the reaction conditions.

Figure 11.In vitro digestion of eGFP plasmid. (A) Lane M, marker. Lane 1, eGFP plasmid. Lane 2, plasmid digested with EcoRI. Lane 3-10 are according to table B. (B) Experiment design to improve the cleavage efficiency.

week4

Figure 12. Improved in vitro digestion of eGFP plasmid. (A,B) Lane M, marker. Lane 1，eGFP plasmid. Lane 2, plasmid digested with EcoRI. Lane 3-4, sgRNA:Cas9:DNA=10:10:1. Lane 5-6, sgRNA:Cas9:DNA=10:20:1. Lane 7-8, sgRNA:Cas9:DNA=20:20:1. As Figure 12 shows, sgRNA:Cas9=1:2(mole number) has the better gene editing efficiency, and the percentage of cleavage may be higher when the quantity of DNA was reduced. And we also found that the DNA might be tracked in the sample holes due to Cas9 protein.

About Targeting mitochondrion

We learned about how to design sgRNA and molecular cloning experiments, intensively studied the literatures on mitochondrial editing and researched on the process of label entering mitochondrial and in-line granule labeling. At the same time, we searched for various genes on mtDNA, reviewed the literature on mitochondrial diseases, and examined the mutation sites of each gene in mitochondria as well as their effects, sorting out the related materials of mitochondrial genome. Based on the comprehensive research on the existing mitochondrial editing methods, we designed a complete experimental scheme, including the plasmid construction scheme and finished designing primers.

September

week1

COS7-GFP cell Lane was offered by School of Public Health, University of South China.[1] We immediately began to culture cells after receiving them and successfully observed the green fluorescence.

week2

We explored and improved the experimental conditions for many times and finally got a better cutting condition. Then we started to study the cutting efficiency of the system with carrier.

week3

Figure 13.Characterization of sgRNA/Cas9 complex(RNP), BODIPY/RNP, Liposome/RNP, and BODIPY/Liposome/RNP. (A) Z-Ave of sgRNA, Cas9, RNP, Liposome/RNP, BODIPY/RNP, and BODIPY/Liposome/RNP. (B) Zeta potential of sgRNA,Cas9,RNP,BODIPY,BODIPY/RNP, and BODIPY/Liposome/RNP.

week4

Figure 14.In vitro digestion of DNA with BODIPY/RNP. (A) Lane 1, eGFP plasmid. Lane 2-7 are set according to table B. Lane M, marker. (B) Experiment design.

About Targeting mitochondrion

The original fragment was synthesized by PCR and then overlapped.
The first batch of competent cells were made and transformed with the original plasmids to obtain the cloning bacteria, from which we successfully extracted the plasmids.
We doubled digested the plasmids repeatedly with Xba1 and Bmgb1 enzymes. After lots of failures, we did a series of double enzyme digestion pre-experiment to improve the original schemes.

Figure 15.Extraction of pET-NLS-Cas9-6xHis plasmid. Lane M, marker. Lane 1, plasmid.

Figure 16.The result of pET-NLS-Cas9-6xHis plasmid digestion. Lane M, marker. Lane1-2, double digestion with Xba1+Bmgb1.Lane 3, single digestion with Xba1. Lane 4, single digestion with Bmgb1. Lane 5, plasmid.

We change the enzymes to Xba1 and nhe1 endonuclease, redesign primers, extend the insert and complete overlap and amplification of the product. After double enzyme digestion, the vector was connected to insert and transformed into cell. However, we found the final construction in July contain so much mutations in the vector that it can’t be used, we reconverted the original plasmid and extracted the original plasmid to do reconstruction.

Figure 17.The result of pET-NLS-Cas9-6xHis plasmid digestion. (A) cleavage 15mins. Lane M, marker. Lane 1, 1μg plasmid double digestion with Xba1 and Nhe1. Lane 2, 2μg plasmid double digestion with Xba1 and Nhe1. Lane 3, 1μg plasmid single digestion with Xba1. Lane 4, 1μg plasmid single digestion with Nhe1. Lane 5, plasmid. (B) cleavage 60mins. Lane M, marker. Lane 1, 1μg plasmid double digestion with Xba1 and Nhe1. Lane 2, 2μg plasmid double digestion with Xba1 and Nhe1. Lane 3, 1μg plasmid single digestion with Xba1. Lane 4, 1μg plasmid single digestion with Nhe1. Lane 5, plasmid.

Figure 18.Amplification of the overlapped COX8a and Cas9. Lane M, marker. Lane 1, COX8a+Cas9 fragment after amplification. The concentration of gel extraction product is 10.8ng/μl，the volume is 200μl

Figure 19. Double digestion of COX8a+Cas9 fragment. Lane M, marker. Lane 1, COX8a+Cas9 fragment double digestion with Xba1 and Nhe1.

Figure 20.The result of fragments synthetic. Lane M, marker. Lane 1, COX8a fragment. Lane 2, Cas9 fragment. Lane 3, COX8a+Cas9 fragment.

October

week1

Figure 21. EGFP gene disruption of COS7-GFP cell line.

About Targeting mitochondrion

Since the previous clones were all negative, we analysed the cause of failure and redesigned the experimental protocol. The Cox8a fragment was reamplified and digested by double enzymes. Also we explored the conditions of dephosphorylation after double enzyme digestion adn redesign the primers to synthesize the other two plasmids. After repeated vector enzyme digestion and dephosphorylation, fragment amplification and double enzyme digestion and connection as well as ligation and transformation time after time, we finally obtained the cloning bacteria which were proved positive by colony PCR.

Figure 22.The result of pET-NLS-Cas9-6xHis plasmid digestion. Lane M, marker. Lane 1-8, plasmid double digestion with Xba1 and Nhe1. Lane 9, plasmid single digestion with Xba1. Lane 10, plasmid single digestion with Xba1. Lane 11, plasmid.

Figure 23.The result of bacterial colony PCR confirmation. Lane M, marker. Lane1-4, SOD2. Lane 5-13, COX8A. Lane 14-17, ATP5.

Figure 24.The result of construction. (A) The construction of pET-NLS-Cas9-6xHis plasmid with SOD2 MTS. Lane 1, MTS fragment. Lane 2, segment of Cas9. Lane 3, overlap of MTS and Cas9 fragments. Lane 4, re-constructed plasmid. (B) The construction of pET-NLS-Cas9-6xHis plasmid with ATP5 MTS. Lane 1, MTS fragment. Lane 2, segment of Cas9. Lane 3, overlap of MTS and Cas9 fragments. Lane 4, re-constructed plasmid.