Team:LZU-CHINA/Proof Of Concept

Part.1 Part.2 Part.3 Part.4 Part.5 Part.6

We firstly verified the specific cleavage activity of CRISPR-Cas13d system targeting ACE2 by “SHERLOCK”. Besides, the CRISPR-Cas13d system targeting ACE2 was designed and packaged into lentivirus. Then we detected the influence of it on cell proliferation and apoptosis. After development of ACE2-293T stable cell line and preparation of SARS-CoV-2 pseudovirus, the inhibitory effect of CRISPR-Cas13d system on SARS-CoV-2 infection was successfully determined.

1. Constructed HEK293T-hACE2 stable cell line

1.1 Expression of RfxCas13d protein

1.1.1 Construction of expression vector

Firstly, Cas13d gene and pET-28a vector were amplified by PCR and purified by agarose gel electrophoresis.
Fig.1. Agarose gel electrophoresis result of Cas13d and pET-28a PCR products
The products were digested with restriction endonucleases Hind III and Bam HI, respectively, and then purified by agarose gel electrophoresis (Fig.2).
Fig.2. Agarose gel electrophoresis result of double digestion
We used T4 DNA ligase to connect Cas13d gene and linearized pET-28a vector. The products were introduced into competent cells of E.coli by the competent cell method, and the plate containing kanamycin was coated (Fig.3). After the colony grow, the colony PCR was used to verify (Fig.3). Further sequencing verification was carried out for the correct colony expansion culture.
Fig.3. Plate coating and colony PCR. Colony 3 is the right connection

1.1.2 Extended culture of Rosetta with recombinant plasmids

The recombinant plasmid pET28-RfxCad13d was transformed into Rosetta. The bacterial solution was inoculated in the medium containing kanamycin and chloramphenicol. Rosetta with empty pET-28a vector was used as contral. Both were induced by IPTG. A group without IPTG was also prepared. The bacteria were centrifuged and collected. We discarded the supernatant and saved the bacteria.

1.1.3 Validation of the expression of RfxCad13d

Bacteria were resuspended. The bacterial liquid was broken by ultrasonic cell disruption instrument (Biosafer, 650-92). The supernatant and precipitation of cell disruption liquid were collected. We collected the supernatants and precipitates of the control and experimental groups for SDS-PAGE. After electrical transfer, the protein samples treated by gel electrophoresis were stained with primary antibody (proteintech, 66005-1-lg) and secondary antibody (proteintech, SA00001-1). We used Western Blot to test the expression of RfxCad13d.
Fig.4. Western Blot results
1.empty vector, supernatant 2.empty vector, precipitation 3.uninduced, supernatant 4.uninduced, precipitation 6.inducted, supernatant 7.inducted, precipitation 8.inducted, supernatant 9.Inducted, precipitation
We can clearly observe the expression of Cas13d protein (Fig.4).

1.1.4 Purification and concentration of protein

Since the target protein of RfxCad13d contains His-Tag, nickel column chromatography was used to purified the protein. We added the eluent to the protein ultrafiltration tube (Pall Corporation, 30K) , after centrifugation，we got RfxCad13d protein concentrate.

1.2 Target RNA preparation

The target RNA（mRNA of ACE2）used in this project was prepared by in vitro transcription using T7 RNA polymerase. Concisely, the double-stranded DNA (dsDNA) carrying T7 promoter sequence were prepared by PCR. Then the transcription reaction was carried out with DNA templates using the T7 High-Efficiency Transcription Kit (TransGen Biotech) at 37°C (1 h). The RNA was purified by the Easy Pure RNA Purification Kit (TransGen Biotech) and quantified with Nanodrop. Then purified RNA’s quality was assayed by nondenaturing agarose gel electrophoresis (Fig.5).
Fig.5. Purified products of in virto transcription on nondenaturing agarose gel

1.3 Test of the specific cleavage activity of RfxCas13d

Once the Cas13d-crRNA complex specifically binding to the target ssRNA, the trans-cleavage activity of Cas13d will be turned on. The collateral RNase activity of Cas13d results in indiscriminate cleavage of the designed short ssRNA reporter (a 6 nt ssRNA modified with 5′-FAM and 3′-BHQ1), generating amplified fluorescence signals. Used ABI Step one plus real-time PCR to detect the fluorescence. We tested the kinetic curve of the protein to see the dynamic change of the flourescence. Preformed complexes of Cas13d and sgRNA were incubated with ssRNA reporter in the presence or absence of target RNA and high fluorescent signal of the specific trans-cleavage activity was observed for RfxCas13d only in the presence of target RNA sample (Fig.6).
Fig.6. Cleavage activity of CRISPR-Cas13d system targeting ACE2 mRNA
(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)

2. Development of ACE2-293T stable cell line

2.1 Construction of recombinant plasmid pLenti-hACE2-T2A-mRFP-puro

The recombinant plasmid pLenti-hACE2-T2A-mRFP-puro was constructed and transformed in E.coli Stbl3, and the plasmid was extracted by endotoxin-free plasmid extraction kit.
The pLenti-puro vector plasmid was digested with endonucleases( XbaI&SpeI )to construct the linearized vector. After agarose gel electrophoresis, specific DNA bands were recovered by gel recovery kit.
Fig.7. Agarose gel electrophoresis result of double digestion
ACE2 (2445 bp) and RFP (678 bp) were amplified by PCR, and the end of ACE2 gene and the beginning of RFP were added with parts of sequence from T2A gene.
Fig.8. Agarose gel electrophoresis result of ACE2 and RFP PCR
Then we use Overlap PCR to connect them together.
Fig.9. Agarose gel electrophoresis result of Overlap PCR
The target band was selected and the specific DNA band was excised. The linearized vector obtained in the previous experiments was ligated with the hACE2-T2A-mRFP gene fragment. Recombinant plasmid transformed into competent cells E. coli Stbl3. After overnight culture, the bacterial colonies plate were picked for colony PCR.
Fig.10. Plate coating of recombinant plasmids transformed into competent cells Restriction digest was adopted to test the vector constructed.
Fig.11. Restriction digest test result. Lane1 is undigested plasmid; Lane2 is plasmid digested with AflII
We also constructed pLenti-hACE2-puro plasmid, which is a simpler vector without T2A and ACE2. It can also be used to establish ACE2 stable cell line.
Fig.12. Map of pLenti-hACE2-PURO

2.2 Construction and screen of ACE2-293T stable cell line

Then recombinant plasmid was transfected into 293T cells by lipo2000 together with pMD2.G and PAX2. After screening with puromycin, viable monoclonals were selected. After expanded culture, the high expression level of ACE2 is determined by qPCR and Western-Blot (Fig.13 and Fig.14).
Fig.13. Expression of ACE2 in stable cell line by qPCR
Fig.14. Expression of ACE2 in stable cell line by Western-Blot

3. Preparation of SARS-CoV-2 pseudovirus

3.1 Virus packaging and WB test

We got a lentiviral plasmid with SARS-CoV-2 spike protein gene on it from addgene. It was co-transfected with PAX2 and a lentiviral plasmid with GFP into 293T cells. The spike protein of SARS-CoV-2 in cell and medium was detected by Western-Blot. HEK293T cells were transfected for 40 h. RIPA lysis buffer was used to lyse cells. pMD2.G plasmid is used as control.
The samples were separated by SDS-PAGE and transferred to PVDF membrane for Western Blot detection. The membrane was incubated with primary antibody and secondary antibody. We observed the result after using ECL chemiluminescence reagent. (Fig.15)
Fig.15. SARS-CoV-2 spike protein expression in cell and medium

3.2 Test of the pseudovirus infection effect

We added the medium containing pseudovirus to ACE2-293T stable cell line. After 48h, fluorescence microscope was used to observe the infection effect. As the result shows, pseudovirus is prepared successfully.
Fig.16. Infection effect of SARS-CoV-2 pseudovirus

4. Design of the CRISPR-Cas13d system targeting ACE2

Three spacer targeting ACE2 mRNA were screened, and the CRISPR-Cas13d system (Fig.17) targeting ACE2 mRNA was constructed by direct repeat tandem expression and connecting with Cas13d gene, which was placed in lentivirus plasmid. Three plasmids were co-transfected into 293T cells to collect viruses, and the viruses are used to knock down ACE2 on the cell surface.
Fig.17. CRISPR-Cas13d system targeting ACE2 mRNA

5. Cell proliferation and apoptosis detection

We detected whether CRISPR-Cas13d system does harm to cell proliferation and apoptosis after knocking down. CRISPR-Cas13d system was added to the ACE2-293T cells. CCK-8 kit was used to detect cell proliferation activity. The result shows the cell proliferation of ACE2 knowdown group cells is slightly lower than the control group and they are not significantly different (P value is 0.1197).
Fig.18. Cell proliferation detection result. OD450 is directly proportional to cell proliferation and reflects cell proliferation activity
Fig.19. Cell apoptosis detection result
FITC-Annexin V/PI cell apoptosis kit was used to detect cell apoptosis. The established stable line cells 293T/hACE2 were cultured in a six-well plate. The lentivirus was added in the experimental group to knockdown ACE2 expression level, while no treatment was done in the control group. After 2 days, one-well samples were stained with FITC-Annexin V and PI for compensatory regulation according to the instructions of FITC-Annexin V/PI apoptosis assay kit. Subsequently, all samples were detected by flow cytometry (LSRFortessa, BD, USA). Although the numbers of apoptotic cells were slightly increased in the experimental group compared with control group, the number of necrotic cells was decreased and the number of living cells was almost the same as that in the control group, suggesting that the knockdown of ACE2 receptor hardly causes negative effect on 293T cells.

6. Determination of the inhibitory effect of CRISPR-Cas13d system on the infection of SARS-CoV-2 pseudovirus

Fig.20. Analysis of CRISPR-Cas13d system targeting ACE2 on the infection of SARS-CoV-2 pseudovirus
We used the established stable line cells ACE2-293T, which routinely cultured in DMEM medium containing 10% fetal bovine serum (including 1% penicillin and streptomycin) in a saturated humidity incubator at 37℃ and 5% CO2. The medium was changed every 2 days. After overnight passage in a six-well plate, the experimental group was added with lentivirus carrying CRISPR-Cas13d system for ACE2 knockdown. After 48 hours of treatment, both the control group and experimental group were added with pseudovirus carrying GFP to simulate SARS-CoV-2 infection. After co-incubating more than 48 hours, the fluorescence intensity of all samples was measured by flow cytometry (LSRFortessa, BD, USA). The results exhibited that the knockdown of ACE2 receptor decreased the infection level of pseudovirus in cells.