Figure 3.

CRISPR/Cas9 Mediates Disruption of APP^SW or APP^WT Allele in Patient-Derived Fibroblasts

(A and B) Sanger sequencing of the APP gene from mutation carrier No. 1 and control line No. 1 transfected with Cas9-2A-GFP and gRNA targeting the APP^SW (SW1, SW2, and SW3) or APP^WT allele (WT), respectively. GFP-expressing cells were sorted 3 days after transfection. On the APP^SW/WT cell line (A), SW1, SW2, and WT gRNA were disruptive and created indels (as indicated by background peaks [i.e., heterogeneity in reads] downstream of the cut site, black arrows). On the APP^WT/WT cell line (B), WT gRNA led to indel formation, whereas SW1, SW2, or SW3 gRNA did not. PAM, protospacer-adjacent motif. The purple arrow shows the predicted CRISPR cut site. (C) Targeted deep sequencing of the APP allele from CRISPR-treated APP^SW/WT fibroblasts (top pie charts) and control APP^WT/WT fibroblasts (bottom pie charts) is shown. Colors indicate mutant (red), wild-type (green), mutant with indels (light red), and wild-type with indels (light green) reads. For some reads, we were not able to determine their origins as mutant or wild-type (due to sequencing errors or larger deletions), and these are marked as white areas on the pie charts. SW1 and SW2 gRNA resulted in indel formation in the APP^SW, but not in the APP^WT allele. WT gRNA resulted in indel formation only in the APP^WT allele in APP^SW/WT cells and APP^WT/WT cells. SW3 did not lead to indel formation in any of the alleles. (D) Coverage of the APP allele in APP^SW/WT cells, treated with Cas9 and SW1 gRNA, is shown. Deletions were detectable adjacent to the CRISPR cut site on the APP^SW allele (red trace). No deletions were present on the APP^WT allele. The PAM site is marked in red. (E) The most frequent reads in the APP^SW/WT fibroblasts treated with Cas9 and SW1 gRNA are shown. PAM sites are marked with red, and the APPswe mutation is marked in yellow. The percentages refer to the fraction of reads that contained any indels.