CRISPR-mediated generation of the APLD humanized transgenic mouse. (A) Three-dimensional structure of the GPIIIa PSI domain, showing the location of the residues that were mutated in the murine protein to humanize the 22 to 40 amino acid loop. (B) Schematic illustration of the ITGB3 locus, showing the location of the gRNA binding site (red bar), the protospacer adjacent motif (PAM) sequence (magenta bar), and the Cas9 cleavage site (red arrow heads). A 200-bp APLD homology directed repair (HDR) template was designed to introduce the 4 desired amino acid substitutions (mutated nucleotides labeled in red) and a diagnostic BamH1 restriction site (silent mutation nucleotides labeled in blue) flanked by 80 nucleotide homology arms. The HDR template also introduces nucleotides (green) that encode silent mutations to prevent recleavage by Cas9. (C) The 20-bp gRNA shown in panel B, designed to target the Cas9 nuclease to the ITGB3 gene, was cloned into the BbsI site of the CRISPR vector px459, which also encodes both Cas9 and a puromycin-resistance gene. Pronuclei of C57BL/6N fertilized eggs were microinjected with the px459 plasmid along with the HDR template to generate the humanized APLD mouse. (D) PCR strategy designed to report the incorporation of the HDR template within a 717-bp region surrounding the targeted site of the murine ITGB3 gene. The introduced BamH1 is marked by a blue box. (E) Genotyping of 2 representative pups. Genomic DNA from the pups’ tails was PCR amplified and digested with BamH1 to identify correctly targeted APLD alleles. The PCR product of pup 1 cut with BamH1, demonstrating successful incorporation of the HDR oligo. The arrows indicate the expected BamH1 digestion products. (F) The ITGB3 locus surrounding the genomic editing site was PCR amplified from genomic DNA of pup 1 and subjected to DNA sequence analysis, confirming precise homozygous integration of the human sequence into both alleles of murine ITGB3.