Part:BBa_K3760002

EF-1α promoter+RfxCas13d+NLS

Design

To express Rfx Cas13d protein in human cell line and cleavage some coding RNA.We fused an EF-1α core promoter(BBa_K1119004) to the 5'end of RfxCas13d coding sequence .Added a bipartite nuclear localization signal from nucleoplasmin(BBa_K2872032) to the 3'end of RfxCas13d coding sequence. To knock down the expression of ACE2, BBa_K3760002 and BBa_K3760011 are used to construct the plasmid, pLenti-sgRNAs-RfxCas13d-PURO.This 3rd generation lentiviral plasmid loads the CRISPR-Cas13d system targeting ACE2 mRNA.
Three spacer targeting ACE2 mRNA are screened, and the CRISPR-Cas13d system targeting ACE2 mRNA is constructed by direct repeat tandem expression and connecting with Cas13d gene, which was placed in lentivirus plasmid. Three plasmids are co-transfected into 293T cells to collect viruses, and the viruses are used to knock down ACE2 on the cell surface.

Figure 1 CRISPR-Cas13d system targeting ACE2 mRNA

Result

1. 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).

Figure 2 Cell proliferation detection result.OD450 is directly proportional to cell proliferation and reflects cell proliferation activity

Figure 3 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.

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

Figure 4 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 ACE2. After 48 hours of treatment, both the control group and experimental group were added with pseudo virus 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 pseudo virus in cells.

Reference

[1]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. Published 2021 Jun 11. doi:10.3389/fcell.2021.667879.
[2]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 Jun;38(6):722-727. doi: 10.1038/s41587-020-0456-9. Epub 2020 Mar 16. PMID: 32518401; PMCID: PMC7294996.
[3]Gurley SB, Allred A, Le TH, et al. Altered blood pressure responses and normal cardiac phenotype in ACE2-null mice. J Clin Invest. 2006;116(8):2218-2225. doi:10.1172/JCI16980

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3164
  Illegal XhoI site found at 2586
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 399
  Illegal NgoMIV site found at 3121
  Illegal NgoMIV site found at 3140
  Illegal AgeI site found at 84
  
• 1000
  COMPATIBLE WITH RFC[1000]