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Fig. 5 Examples of HDR template design to knock-in a point mutation. (A) The point mutation G>A (shown in blue font) modifies the PAM site as well as generates an AluI restriction site that can be used for screening. (B) The point mutation G>C (shown in blue font) is 17 bp downstream of the DSB site. A silent change is made to disrupt the PAM site (G>C, shown in magenta font) and this change also generates a SalI restriction site that can be used for screening. (C) The point mutation C>A (shown in blue font) is 6 bp upstream of the DSB site. In this example, a silent change cannot be made to the PAM site, and, therefore, four silent changes are required (shown in magenta font) to destroy the sgRNA binding site. One of these changes also generates a SfcI restriction site that can be used for screening. In all examples, the DSB site is denoted by a red arrowhead, sgRNA is shown in bold font, and the PAM site is underlined.