Team:LZU-CHINA/Engineering

Part.1 Part.2 Part.3 Part.4

1. Constructed HEK293T-hACE2 stable cell line

To construct SARS-CoV-2 pseudovirus’ target cell, first we constructed the transfer plasmid pLENTI-ACE2-PURO in virto. We then introduced it with packaging plasmids into HEK293T cells. Then we screened HEK293T-hACE2 stable cell line using purinomycin, but we have difficulty in screening. To screen the stable cell line easier, we designed a part BBa_K3760004 which contains T2A and mRFP to replace ACE2 coding sequence in pLENTI-ACE2-PURO.
Fig.1. (a)pLENTI-ACE2-PURO; (b)The Western Blot of ACE2

2. Constructed the SARS-CoV-2 pseudovirus

We transfected pCMV14-3X-Flag-SARS-CoV-2 S, pLenti-CMV-GFP-Puro (658-5), psPAX2 into HEK293T cells and packaged the pseudovirus. Then We harvested the virus suspension and stored at -80°C. We also used Western Blot to make sure the Spike protein was expressed.
Fig.2. The Western Blot result of Spilke protein

3. Constructed and characterized a new part：BBa_K3760001 6x His tag-RfxCas13d

3.1 Cloning

We ordered RfxCas13d gene from Addgene, then we cloned the gene into pET-28a. Additionally, we attached a 6 x His tag to 5' end of the gene. We transformed the ligation product into E.coli Rosetta (DE3) and confirmed the cloning by colony-PCR and sequencing.
Fig.3. Colony PCR result of 6xHis RfxCas13d in E.coli Rosetta. Note: Lane 3 is the positive clone

3.2 RfxCas13d protein expression and purification

We expressed the protein in E.coli Rosetta (DE3). Expressed it at 16°C in LB medium under the condition of 1 mM IPTG. And as the sequence contains 6×His tags, we purified the protein through Ni-NTA agarose.
Fig.4. The RfxCas13d purification result. 1.empty vector supernatant 2.empty vector precipitation 3.uninduced supernatant 4.uninduced precipitation 6.Inducted supernatant 7.Inducted precipitation 1 8.Inducted supernatant 9.Inducted precipitation

3.3 Characterization: Test the cleavage activity of RfxCas13d protein

After we got the expressed and purified RfxCas13d protein, we needed to verify its activity to make sure our CRISPR-Cas13d system can target the coding RNA of ACE2. We tested the kinetic curve of the protein to see the dynamic change of the flourescence. We can clearly see that after adding the target, the activity of the protein is greatly improved, which enables us to tell whether the coding RNA of ACE2 was splited. After 100 mins of detecting, the fluorescence signal gradually became stable.
We analysed it on the model page, click >>Model<< to see more details.
Fig.5. Cleavage activity of RfxCas13d in virto.(a)The fluorescence signals of target RNA(A,B,C) and control(D,E,F,G) were detected on real-time PCR.(b) The cleavage activity of RfxCas13d were tested with ssRNA reporter. Control: Without target RNA. Error bars represent the means ± SD from three replicates (n = 3)

4. Constructed the lentivirus carried CRISPR-Cas13d system to knock down ACE2

We constructed the plasmid pLenti-sgRNAs-RfxCas13d-PURO to express RfxCas13d and sgRNAs which is desgined to knock down the expression of ACE2 in HEK293T-ACE2 cells. This vector and two packaging plasmids were co-transfected into cells.
Fig.6. pLenti-sgRNAs-RfxCas13d-PURO
Thorugh RT-PCR, we detected that the expression level of ACE2 has degraded significantly. In the simulated infection of SARS-CoV-2 pseudovirus, we used flow cytometry (LSRFortessa, BD, USA) to measure the fluorescence intensity of all samples. The results exhibited that the knockdown of ACE2 receptor decreased the infection level of pseudovirus in cells. This system can prevent HEK293T-ACE2 cells from the infection of SARS-CoV-2 pseudovirus.
Fig.7. Analysis of CRISPR-Cas13d system targeting ACE2 on the infection of SARS-CoV-2 pseudovirus