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. 2018 Nov 16;8:16945. doi: 10.1038/s41598-018-35079-y

Figure 1.

Figure 1

Locus of HPP mutation targeted by sgRNA/Cas9 and targeting efficiency in the ALPL sheep gene. (A) Schematic of exon 10 (red) with the locus of the HPP c.1077 for the cytosine to guanine (C > G) point mutation (purple). The PAM sequence is in orange where an additional silent point mutation was created to limit Cas9 cleavage. (B) Detection of sgRNA/Cas9 targeting efficiency in the ALPL gene analyzed with T7E1 assay in sheep fibroblasts. Arrows indicate expected cleaved 600 bp and 400 bp fragment sizes of target regions with sgRNA#1, sgRNA#2, and WT control. (C) PCR detection of sgRNA/Cas9 targeting modifications in newborn lambs, individually numbered at the bottom. PCR product (959 bp) flanking target region from DNA extracted from individual newborn lamb umbilicus. (D) Summary of genetically-engineered lambs targeting the ALPL gene with CRISPR/Cas9. IVC, in vitro cultured.