Figure 6.

Design and selection of sgRNAs for efficient targeting of the pNGF gene via the CRISPR/Cas9 system. (A) Location of the recognition sites of 11 designed sgRNAs that target the pNGF gene. (B) The map of the CRISPR/Cas9 system expression plasmid (ppNGF-sgRNA-Cas9) that targets the pNGF gene. (C) Ear fibroblasts isolated from no. 5 F₀ hNGF-TG pig for the second round of genetic modification. (D) T7E1 assay of the targeting efficiency of 11 designed sgRNAs. 1–11: the ID no. of 11 designed pNGF-targeting sgRNAs. (E) Analysis of the targeting efficiency of sgRNA5 and sgRNA10 by sequencing the PCR amplification products of their targeting sites following transfection of the ppNGF-sgRNA5-Cas9 and ppNGF-sgRNA10-Cas9 plasmids into pig fibroblasts. TE, targeting efficiency.