Fig. 1: Schematics of the genome editing tools and assessment of cellular nuclease distribution. | Gene Therapy

Fig. 1: Schematics of the genome editing tools and assessment of cellular nuclease distribution.

From: Integrating gene delivery and gene-editing technologies by adenoviral vector transfer of optimized CRISPR-Cas9 components

Fig. 1

a Schematic representation of the constructs deployed to express the CRISPR-Cas9 components. The hybrid CAG promoter is composed of sequences from the cytomegalovirus (CMV) immediate-early enhancer, the first exon/intron of the chicken β-actin gene, and the splice acceptor of the rabbit β-globin gene. Magenta stripes, nuclear localization signal (NLS) from the SV40 large T antigen; cyan stripes, NLS from the nucleoplasmin of Xenopus sp.; rBGpA, rabbit β-globin polyadenylation signal; U6, RNA Pol-III promoter from the human U6 gene; opt-gRNA, gRNA with an optimized tracrRNA; red stripes, nt differences between conventional gRNA and opt-gRNA scaffolds. b SpCas9 immunofluorescence microscopy on HeLa cells. HeLa cells transfected with the indicated combinations of constructs were stained for SpCas9. Two representative fluorescence microscopy images for each experimental condition were acquired at 5 days post transfection. The staining for SpCas9 proteins and DNA was performed with an antibody specific for the C-terminus of SpCas9 and DAPI, respectively. The white horizontal bar corresponds to 30 µm.

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