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Fig. 2 | Genome Biology

Fig. 2

From: CRISPR/Cas9-mediated genome editing induces exon skipping by alternative splicing or exon deletion

Fig. 2

Ctnnb1 sgRNAs targeting exon 3 induces exon skipping. a Schematic of the Ctnnb1 gene. The in-frame exon 3 encodes an inhibitory domain: phosphorylation amino acids 33, 37, 41, and 45 promotes degradation of the β-Catenin protein. Loss of exon 3 stabilizes β-Catenin. Eleven sgRNAs were designed to target exon 3: strong sgRNAs in red and weak sgRNAs in black, respectively. sgRNAs that use “NGG” PAM are shown above exon 3 and those that use “CCN” PAM are shown below exon 3. b Correlation between exon 3 skipping and sgRNA efficiency. Genomic indels were measured by deep sequencing. KP cells were infected with lentivirus. Exon 3 skipping efficiencies are from (d). Indels of sg11 were not determined. sgRNAs that induce > 20% indels are marked in red. c Distribution of sg1 indels shows that a T insertion (+T) at the Cas9 cleavage site nucleotide 97 of exon 3 (red arrowhead) was the most frequent. PAM sequence is in blue. d RT-PCR using primers spanning exons 2 and 5 shows partial exon skipping. M molecular marker. sgGFP is a control sgRNA. Exon 3 skipping bands were quantified using ImageQuant TL software and normalized to full length cDNA bands. sg4 showed visible weak bands that could not be quantified. e, f TOPO cloning and Sanger sequencing confirmed that the two major lower RT-PCR bands in (c) are alternative splicing of exon 2-4 and exon 2-5, respectively. g Western blot analysis of β-Catenin. Full length β-Catenin is ~86 kD. β-Catenin without exon 3 (delta exon 3) is ~77 kDa. Actin served as a loading control

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