Establishing the type 2N VWD mouse model using a CRISPR/Cas9 strategy. (A) A gRNA and asymmetric single-stranded oligodeoxynucleotide homology-directed repair template spanning the predicted Cas9 cleavage site were designed to introduce 3 single nucleotide changes into the mouse Vwf gene. The 2354G>a [G785E] mutation causes type 2N VWD. The other 2 changes are silent mutations, 2346G>a and 2358G>c, which eliminate the Cas9-requisite protospacer adjacent motif sequence and create a diagnostic Xho I site for genotyping, respectively. (B) Schematic diagram of genotyping by PCR and Xho I restriction enzyme digestion. (C) PCR detection of the 2354G>a variant mouse Vwf gene for genotyping. DNA was purified from peripheral blood leukocytes, and a 796-bp fragment was amplified from the mouse Vwf gene. PCR product was digested with Xho I and run on 1.25% gel. A single uncut fragment of 796 bp is observed for the wild-type mouse Vwf PCR product because it does not contain an Xho I restriction enzyme site. Two fragments (585 and 211 bp) result from the mutated type 2N mouse Vwf gene after Xho I digestion. Thus, a VWF^+/+ genotype results in a single 796-bp band, VWF^2N/2N genotype is indicated by 585- and 211-bp bands, and VWF^2N/+ mice exhibited 796-, 585-, and 211-bp bands.