Figure 1 | Laboratory Investigation

Figure 1

From: Rapid generation of Col7a1−/− mouse model of recessive dystrophic epidermolysis bullosa and partial rescue via immunosuppressive dermal mesenchymal stem cells

Figure 1

CRISPR/Cas9-based disruption of type VII collagen by embryo injection. (a) Strategy using the CRISPR/Cas9 nuclease system to produce Col7a1−/− NSG mice. CRISPR guide RNA and Cas9 mRNA are injected into cytoplasm of single-cell NSG embryos, which are then transferred to CD-1 pseudo-pregnant female surrogates. Upon birth, visibly blistered animals were used for transplantation and/or survival experiments, whereas the non-blistered animals were kept for genotyping and subsequent breeding colony establishment. (b) First coding exon of murine Col7a1. To maximize the frequency of frameshift mutations, two guide RNAs (gRNAs) (green text) were designed to cut near each other within first coding exon. Start codon in red, PAM sequences in orange. Cut site indicated by red triangle. (c) Surveyor assay from transient transfection used to validate the nuclease assay of each gRNA. Red triangles indicate the Surveyor cleavage products.

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