Figure 2
Generation of a Gaaem1935C>A transgenic mouse line. (A) Dual overlapping guide RNA approach targeting the Gaac.1935 target locus. Arrowhead direction indicates whether guide RNA is sense (right) or antisense (left). PAM sequences (NGG) are highlighted in color corresponding to the guide RNA arrows. The Gaac.1935 locus for target adenine to cytosine nucelotide transversion is highlighted in red. Expected Cas9 nuclease cut sites are shown as vertical arrows in color corresponding to the guide RNA arrows. (B) Sequence of the target locus for integration (top) aligned with the ssODN (bottom) to introduce the Gaac1935C>A mutation (green box). PAM motifs are indicated in gold (gRNA-2) or blue (gRNA-3). Installed synonymous variants at PAM sites (Gaac.1920C>T, Gaac.1932G>A) and the desired KI mutation are highlighted in red. Installed gRNA seed region variants (Gaac.1923G>C, Gaac.1929G>A) are highlighted in green. (C) Sequencing chromatograms of control (Gaawt), founder #1 (Gaac.1935 Founder #1), and founder #2 (Gaac.1935 Founder #2). Black arrows indicate synonymous variant edits at PAM sites (Gaac.1920, Gaac.1932) or gRNA seed regions (Gaac.1923, Gaac.1929). Red arrows indicate the desired KI mutation (Gaac.1935C>A). Gray shaded region indicates amino acids at position 645 for each mouse. (D) WGS analysis (> 50 × read depth) of the Gaac.1935 locus in G0 founder #1 and G0 founder #2. WGS analysis demonstrates highly efficient on-target genome-editing in these founder mice. Data are presented as stacked bar graphs indicating the percentage of WGS reads for each event category. Gray: nonspecific Gaa mutations; black: no Gaa mutation; red: intended Gaac.1935C>A mutation and associated synonymous variants; and blue: Gaa insertion/deletions. (E) Pedigree diagram of mating scheme to segregate the intended Gaaem1935C>A KI allele from mosaic CRISPR-generated founder mice for generation of homozygous Gaaem1935C>A KI mice. Males are represented as squares and females are represented as circles.
