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. 2023 Mar 6;15(3):686. doi: 10.3390/v15030686

Figure 2.

Figure 2

Targeting of SARS-CoV-2 RNA with CRISPR/Cas13d. (A) SARS-CoV-2 RNA genome diversity and the crRNA targets. Distribution of crRNAs along the SARS-CoV-2 RNA genome (MN908947). The Shannon entropy along the RNA genome varies from 0 to 1, where lower genetic diversity gives values closer to zero.The position of the crRNAs targeting conserved RNA sequences of SARS-CoV-2 is indicated with black triangles. The SARS-CoV-2 RNA genome diversity is shown by plotting Shannon entropy along the whole genome. The values vary between 0 and 1, where more conserved sequences give lower values (closer to 0); (B) Luciferase multitarget reporter construct. To measure the efficiency of the designed crRNAs, a pGL3 control plasmid-based multi-target luciferase reporter construct was designed with SARS-CoV-2 conserved target sequences cloned downstream of the firefly luciferase gene. Two constructs were designed: one with the + and one with the −RNA target sequences. SV40: simian virus 40 promoter, pA: polyA sequence; (C) Engineering a SARS-CoV-2 replicon. Scheme of the SARS-CoV-2 cDNA cloned in a BAC (upper panel) and the SARS-CoV-2 replicon (lower panel). The letters indicate the viral genes: S, spike; E, envelope; M, membrane; N, nucleocapsid; or the reporter gene mNeonGreen (mNG). UTR, untranslated regions; CMV, cytomegalovirus promoter; pA, polyA sequence; Rz, hepatitis delta virus ribozyme; BGH, bovine growth hormone polyadenylation and termination signals.