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. 2019 May 23;7(7):e00755. doi: 10.1002/mgg3.755

Figure 3.

Figure 3

CCM1‐/‐ mutant BOECs acquire clonal dominance in co‐culture. (a–d) Sanger sequencing analyses of DNA samples from clonally expanded BOEC colonies after CRISPR/Cas9 genome editing demonstrated (a) the HDR‐corrected sequence with the silent variant c.2013C>T, p.(Asn671=), (b) the in‐frame variant c.[2012delA;2020dupA], p.(Asn671_Glu673delinsThrTrpLys), (c) the patient‐specific one base pair deletion c.2012delA, p.(Asn671Thrfs*36) in heterozygous states and (d) the compound heterozygous knockout alleles c.[2012delA];[2014_2021del], p.[Asn671Thrfs*36];[Met672*]. Nucleotide calls and amino acid changes (single letter code) are given above the electropherograms. Predicted consequences of the nucleotide changes are depicted at the bottom of each subpanel. E = exon, AA = amino acid. (e) CCM1‐/‐ clones display significantly reduced caspase‐3 activity under induction of apoptosis with 1 µM staurosporine. Data are presented as mean and SEM. Two‐way ANOVA was used for statistical analysis: **p < 0.01. (f) Amplicon deep sequencing results for cell mixtures over time. The corrected cell mixture shows a constant allele ratio for 34 days of culture (left) whereas the addition of 10% compound heterozygous CCM1‐deficient cells (c.[2012delA];[2014_2021del] corresponds to c.[2014_2021del] in 5% of all alleles, red color) to the corrected cell mixture leads to a strong shift toward knockout alleles already after 16 days (right)