Strategy used to convert the Pl^A1 allelic form of GPIIIa to Pl^A2. (A) Schematic illustration of the ITGB3 locus, showing the location of the 2 gRNA binding sites (orange bars) and the protospacer adjacent motif (PAM) sequences (magenta) necessary to guide Cas9n to its cleavage site (red arrowheads). A 181 bp Pl^A2 HDR template was designed to introduce the Leu→Pro amino acid polymorphism. The T>C mutation responsible for the Pl^A1/Pl^A2 polymorphism (highlighted in red) is flanked by 90 nucleotide homology arms and creates an NciI site at the target locus that can be used for genotyping.¹³  The HDR template also contains 2 silent mutations (highlighted in blue) to prevent recleavage by Cas9n (see “Materials and methods”). (B) The gRNAs were cloned into the BbsI site of the CRISPR vectors px461 or px462, which encode GFP or a puromycin-resistance gene, respectively, as well as Cas9n. The use of 2 different guides to direct the Cas9n nickase to nearby sites at this locus significantly reduces the incidence of off-target mutations relative to that incurred using a single-guide RNA and the double-strand nuclease Cas9.^27,28