Fig 1: In vivo genome editing decreased CTNNB1 expression. (A) qRT-PCR analysis of Ctnnb1 mRNA expression in Tie2Cas9 mice treated with AAV-BR1-sgCtnnb1-tdTomato. Data are means ± SEM. (n = 4 mice, ** P < 0.01, two-tailed unpaired t-test). (B and C) Western blots (B) and quantification (C) of CTNNB1 levels in brain ECs isolated from edited mice. Data are means ± SEM. (n = 3 mice, ** P < 0.01, two-tailed unpaired t-test). (D) Confocal microscopy images of PLVAP (green), CTNNB1 (gray), tdTomato (red), and CD31 (purple) immunostaining in Tie2Cas9 mice. Scale bar, 10 µm (left), 4 µm (right).
Fig 2: In vivo genome editing of brain ECs using the AAV-BR1-CRISPR system. (A) The strategy used to incorporate the sgCtnnb1 vector into AAV-BR1 and the scheme of study. (B) Results of flow cytometry analysis used to detect CD31+tdTomato+ ECs of Tie2Cas9 mice with (red) or without (blue) AAV-BR1-sgCtnnb1-tdTomato intravenous injection. Data are means ± SEM. (n = 4 mice). (C) T7E1 assay of brain ECs isolated from Tie2Cas9 mice at the targeted locus of Ctnnb1 compared to the control group. Lane I was loaded with a molecular weight marker (100 bp ladder). (D) Classification of amplicon sequencing of brain ECs at the Ctnnb1 locus. (n = 3 mice). (E) Indel phase showing that most indels caused a frameshift. (n = 3 mice). (F) Proportion of sequences containing specific mutation types (insertions or deletions) at individual base pair positions out of all mutated sequences. (n = 3 mice). (G) Summary of the most abundant indels in brain ECs obtained from Tie2Cas9 mice treated with AAV-BR1-sgCtnnb1-tdTomato based on CRISPResso2 analysis. Red rectangles denote inserted sequences, while dashes represent deleted nucleotides. The horizontal dashed line indicates the CRISPR cut site.
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