Figure 1.

Design of the synthetic switch to restrict Cas9 transcription and translation. (A) Plasmids to co-express Cas9, L7Ae and EYFP, with (pK-CLY) or without (pCLY) two repeats of K-turn at the 5′ end. The three genes were linked in tandem by P2A sequences and driven by CMV promoter/enhancer. Cas9 was fused with an SV40 nuclear localization signal (NLS). pA, poly A signal. (B) Plasmids to express the gRNA. pTA-gVgC co-expressed two different gRNAs: gRNA_VEGFA1 targeting human VEGFA1 gene on human chromosome 6 and gRNA_Cas9 targeting the plasmid-borne Cas9 gene. pTA-gV expressed gRNA_VEGFA1 while pTA-gϕ expressed a scramble gRNA (gRNA_ϕ) that targets no sequence on the chromosomes or plasmids. All gRNAs were driven by human U6 promoter. Ter, transcription termination sequence. (C) Schematic illustration of how the synthetic switch self-regulates Cas9 expression in the transcription and translation steps. Co-transfection of pK-CLY and pTA-gVgC into cells would lead to co-transcription of Cas9, L7Ae, EYFP, gRNA_VEGFA1 and gRNA_Cas9. Cas9 is translated and orchestrates with gRNA_VEGFA1 to induce on-target indel mutation at VEGFA1 site while also associating with gRNACas9 to inhibit Cas9 transcription by cleaving the plasmid-borne Cas9 gene. Additionally, L7Ae is translated and in turn binds the K-turn motifs at the 5′ UTR of mRNA to suppress mRNA translation.