Journal: Scientific Reports

Article Title: Genetic engineering a large animal model of human hypophosphatasia in sheep

doi: 10.1038/s41598-018-35079-y

Figure Lengend Snippet: 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.

Article Snippet: Two specific sgRNAs targeting exon 10 were designed using the Benchling web based CRISPR gRNA design tool (Benchling, San Francisco, CA) that would result in minimal off-target effects (Supplemental Table ).

Techniques: Mutagenesis, Sequencing, Control, CRISPR, In Vitro, Cell Culture

Journal: Scientific Reports

Article Title: Genetic engineering a large animal model of human hypophosphatasia in sheep

doi: 10.1038/s41598-018-35079-y

Figure Lengend Snippet: Genotype of ALPL c.1077 C > G targeted lambs with CRISPR/Cas9. ( A ) Representative chromatographs from Sanger sequence analysis of newborn lamb umbilicus tissue. ( B ) Photos of ALPL targeted animals from each genotype at 2 weeks of age.

Article Snippet: Two specific sgRNAs targeting exon 10 were designed using the Benchling web based CRISPR gRNA design tool (Benchling, San Francisco, CA) that would result in minimal off-target effects (Supplemental Table ).

Techniques: CRISPR, Sequencing