Fig. 1: Screening of crRNA targeting the Ttr gene in mouse Hepa1-6 cells.
From: CRISPR–Cas3-based editing for targeted deletions in a mouse model of transthyretin amyloidosis
a, Schematic of the mouse Ttr (ENSMUST00000075312.5) and B4galt6 (ENST00000306851) loci, showing five Cas3-targeting crRNAs (crRNA1–crRNA5) and one Cas9 gRNA (G211). Primer pairs used for conventional PCR amplification are indicated, along with the genomic regions interrogated by ddPCR probes for quantitative assessment. b, Analysis of genome-editing efficiency in Hepa1-6 cells following transfection with pCas3-crRNA or px458-gRNA (for Cas9). PCR amplicons of the Ttr target region were visualized by agarose gel electrophoresis. Appearance of smaller amplicons in Cas3-transfected samples confirms efficient cleavage at the intended target sites. NC, nontransfected control. c, ddPCR-based quantification of deletion efficiency at Ttr exon 2. Transfection with pCas3-cr2 yielded ~60% deletion, significantly higher than other crRNAs. Statistical analysis was performed using a one-way ANOVA with Dunnett’s post hoc test versus pCas3-cr2 (n = 3 independent biological replicates per group (separately seeded and transfected Hepa1-6 cell cultures)). d, ddPCR-based quantification of editing at Ttr exon 4 and off-target effects at B4galt6 exon 9 in pCas3-cr2-transfected cells. A deletion frequency of 30.9% ± 2.2% was detected at Ttr exon 4, while off-target editing at B4galt6 exon 9 remained at 4.4% ± 2.0%. No detectable deletions were observed in px458-G211-treated cells. Statistical test was performed using a one-way ANOVA with Dunnett’s post hoc test (versus NC). Error bars indicate the mean ± s.e.m. (n = 3 independent biological replicates per group (separately seeded and transfected Hepa1-6 cell cultures)).
