We build biobricks for our project. These parts cover all aspects in our project, including Cas13d protein expression and purification, Cas13d’s cleavage activity verification in virto, construction of HEK293T-ACE2 sable cell line and the lentivirus carried CRISPR-Cas13d system to degrade mRNA of ACE2.

Part List

Part Name (Link) Description Type
BBa_K3760000 Rfx Cas13d coding sequence coding
BBa_K3760001 6 X His tag-Rfx Cas13d Composite
BBa_K3760002 EF-1α promoter+RfxCas13d+NLS Composite
BBa_K3760003 17 promoter t hACE target RNA coding sequence Coding
BBa_K3760004 hACE2-T2A-mRFP Composite
BBa_K3760005 RfxCas13d-nACE2-sgRNA1 RNA
BBa_K3760006 RfxCas13d-nACE2-gRNA2 RNA
BBa_K3760007 RfxCas13d-nACE2-sgRNA3 RNA
BBa_K3760008 T7 promorter-sgRNA1-Synthesis Other
BBa_K3760009 T7 promorter-sgRNA2-Synthesis Other
BBa_K3760010 T7 promorter-sgRNA3-Synthesis Other
BBa_K3760011 SgRNA expression cassette Composite

1.Cas13 protein

To knock down the expression of ACE2, we choose RfxCas13d protein as our cleavage effector which is found in Ruminococcus flavefaciens. To obtain this protein with high purity and activity, we designed biobricks to express RfxCas13d protein. Meanwhile, we attached 6×His tag to make it can be purified by Ni-NTA purification.BBa_K3760000, BBa_K3760001[1].

2.ACE2 target sequence

To test the cleavage activity of our CRISPR-Cas13d system in virto, we built BBa_K3760003 which cotains coding sequence of ACE2. It is the template for mRNA of ACE2 in vitro synthesis.

3.sgRNA for RfxCas13d

The sgRNA is an oligonucleotide chain, it can combine with Cas13 protein and guide Cas13 to find the target single-stranded RNA. It consists of two parts, DR sequence and spacer. The DR sequence is determined by Cas13 protein. We built BBa_K3760005, BBa_K3760006, BBa_K3760007 to guide our Cas13d protein to target the mRNA of ACE2.

4.Construction of HEK293T-ACE2 stable cell line

To construct HEK293T stable cell line as target cell in cell experiment, we built BBa_K3760004 which will be used to package lentivirus. This part cotains ACE2, T2A and mRFP.

5.Knockdown of ACE2

To knock down the expression of ACE2, we built BBa_K3760002 and BBa_K3760011 which cotains RfxCas13d and sgRNA expression cassette[2].

[1]Konermann S, Lotfy P, Brideau NJ, Oki J, Shokhirev MN, Hsu PD. Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors. Cell. 2018 Apr 19;173(3):665-676.e14. doi: 10.1016/j.cell.2018.02.033. Epub 2018 Mar 15. PMID: 29551272; PMCID: PMC5910255.
[2]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 Jun 11;9:667879. doi: 10.3389/fcell.2021.667879. PMID: 34178991; PMCID: PMC8226256.