We design a lentivirus carrying CRISPR-Cas13d system to degrade the expression of ACE2 on the ACE2-HEK293T cell surface in order to inhibit the entry of SARS-CoV-2 pseudovirus into cells.

1. Verification of CRISPR-Cas13d system in virto

First of all, we use online crRNA design tool[1] to design Cas13d crRNA targeting ACE2 mRNA and choose three spacers which are best-matched with ACE2 mRNA. To vreify whether the CRISPR-Cas13d system we designed can target encoding RNA of ACE2, we use a method that is similar to Feng Zhang’s “SHERLOCK” in theory[2]. We clone the RfxCas13d gene into expression vector pET-28a and transforme it into E.coli Rosetta(DE3). Then collect cells after culture and IPTG induction. After ultrasonic fragmentation, the RfxCas13d protein is purified by Ni-NTA. Finally, the expression of RfxCas13d protein is verified by Western Blot.
The ACE2 mRNA is prepared through T7 RNA Polymerase transcription in virto. Then ACE2 mRNA, chemically synthesized crRNA and RfxCas13d protein are incubated with RNA reporter (FAM-5’UUUUUU-3’BHQ1). Through collateral cleavage, FAM and BHQ1 quencher are separated. The fluorescence release is detected by real-time PCR instrument.
Fig.1. Verification of CRISPR-RfxCas13d in virto Process diagram[2]

2. Construction of targtet cells

To construct the target cells of SARS-CoV-2 pseudovirus, ACE2 gene is inserted into a third generation lentiviral vector. Then this plasmid, pLENTI-ACE2-PURO with pMD2.G and PAX2 are transfected into HEK293T cells by Lipofectamine 2000. After screening with puromycin, viable monoclonals are selected. The overexpression of ACE2 is determined by qPCR and Western Blot after expanded culture.

3. Preparation of SARS-CoV-2 pseudovirus

Because of safety and forbiding use live SARS-CoV-2, we use safer lentivirus that express Spike protein to replace live virus. Three plasmids including pCMV14-3X-Flag-SARS-CoV-2 S[3], PAX2 and pLenti-CMV-GFP-Puro are transfected into HEK293T cells to collect virus. The pseudovirus contains GFP to observe the infected cells in simulated infection experiment.

4. Knocking down ACE2

In order to knock down ACE2 on the target cell surface, we consturct a lentiviral vector, pLenti-sgRNAs-RfxCas13d-PURO, which contains three verifed tandem-expressed crRNA and RfxCas13d gene[4]. The lentiviral plasmid is transfected into HEK293T cells with packaging plasmids to collect viruses. Then the lentivirus carrying CRISPR-Cas13d system is used to infect ACE2-293T cells. The result of koncking down is determined by qPCR.
Fig.2. pLenti-sgRNAs-RfxCas13d-PURO
After koncking down ACE2 in ACE2-HEK293T cells, it’s necessary to detect whether CRISPR-Cas13d system does harm to cells. We use FITC-Annexin V/PI cell apoptosis kit and CCK-8 kit to detect apoptosis and cell proliferation activity .

5. Simulated infection

Finally, we use SARS-CoV-2 pseudovirus to infect ACE2-HEK293T cells. In order to determine the inhibitory effect of CRISPR-Cas13d system designed by us on the infection of SARS-CoV-2, we compare the virus loads in ACE2-HEK293T cells of the control groups and the experimental groups that knock down ACE2.
Pseudovirus with expression of GFP make it easy to compare the virus loads through observe the fluorescence intensity of control groups and experimental groups.

[1] Wessels HH, Méndez-Mancilla A, Guo X, Legut M, Daniloski Z, Sanjana NE. Massively parallel Cas13 screens reveal principles for guide RNA design. Nat Biotechnol. 2020;38(6):722-727
[2] Qiao X, Gao Y, Li J, Wang Z, Qiao H, Qi H. Sensitive analysis of single nucleotide variation by Cas13d orthologs, EsCas13d and RspCas13d. Biotechnol Bioeng. 2021;118(8):3037-3045.
[3] Ou X, Liu Y, Lei X, et al. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV [published correction appears in Nat Commun. 2021 Apr 1;12(1):2144]. Nat Commun. 2020;11(1):1620.
[4] Chuang YF, Wang PY, Kumar S, Lama S, Lin FL, Liu GS. Methods for in vitro CRISPR/CasRx-Mediated RNA Editing. Front Cell Dev Biol. 2021;9:667879.