Fig. 7

Mosaic disruption of theanxa4gene by CRISPR/Cas9 leads to apoptosis in the developing liver. (a) The CRISPR guide RNA is designed to target a region in Exon 3 of anxa4, which contains a DdeI restriction enzyme site. The red lines indicate the guide RNA target site. Orange arrows indicate the primers for the genotyping PCR. (b)-(d) Effective anxa4 disruption by CRISPR/Cas9 was confirmed by DdeI digestion of the PCR products (b) and by Anxa4 antibody labeling of the floor plate (c) and (d). An uncut band (green arrow in (b)) and the floor plate cells without Anxa4 expression (white arrowheads in (d)) are found only in injected embryos. Yellow arrows point to floor plate (fp) in (c) and (d). (e) and (f′′′) Confocal projections showing that apoptosis, indicated by GFP blebs and cleaved Caspase3 labeling, occurs in a subset of liver cells where Anxa4 expression is disrupted in the CRISPR injected embryos (white arrows) but not in control embryos.