Journal: Nucleic acids research

Article Title: CRISPR-Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells.

doi: 10.1093/nar/gkac049

Figure Lengend Snippet: Figure 1. DNA nanostructure encoding mNeonGreen for human genome integration. (A) Graphical strategy depiction showing folding of a long unstruc- tured ssDNA into a DNA nanostructure for integration into the genome via CRISPR–Cas9-mediated HDR. (B) Schematic of a 2716-base long template encoding mNeonGreen along with regulatory elements and two 100-base homology arms for genome targeting atan intergenic site on human chromosome 9. (C) Cylindrical model and oxDNA simulations of an 18-helix bundle DNA nanostructure show a decrease in end-to-end distance from 108.98 ± 11.22 nm (ssDNA) to 29.33 ± 9.9 nm (18-helix). (D) AFM characterization of the unstructured ssDNA and the 18-helix DNA nanostructure. Scale bar: 100 nm.

Article Snippet: Imaging was performed with ScanAsyst-Air probes at a typical scan rate of around 1 Hz. oxDNA simulations Simulations of four distinct versions of 18-helix DNA nanostructured HDR templates were performed using the coarse-grained model oxDNA (25), including 50% Staples, Only Top, Open and Complex.

Techniques: CRISPR

Journal: Nucleic acids research

Article Title: CRISPR-Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells.

doi: 10.1093/nar/gkac049

Figure Lengend Snippet: Figure 4. Nanostructured DNA comprising a human gene enhances human primary cell HDR compared to unstructured dsDNA. (A) Schematic of knock-in strategy of a 3.5-kb HDR template encoding IL2RA–GFP fusion and mCherry driven by an EF1a promoter. (B) oxDNA simulations and AFM images of four distinct versions of 18-helix DNA nanostructured HDR templates, including 50% Staples, Only Top, Open and Complex. Scale bar: 100 nm. (C) Unstructured ssDNA and 18-helix nanostructure templates show increased knock-in efficiency compared to dsDNA. Error bars represent SDs from duplicate experiments. (D) Live cell count shows that unstructured ssDNA and 18-helix nanostructured templates display lower toxicity compared to dsDNA. Error bars represent SDs from duplicate experiments.

Article Snippet: Imaging was performed with ScanAsyst-Air probes at a typical scan rate of around 1 Hz. oxDNA simulations Simulations of four distinct versions of 18-helix DNA nanostructured HDR templates were performed using the coarse-grained model oxDNA (25), including 50% Staples, Only Top, Open and Complex.

Techniques: Knock-In, Cell Counting