Bellchambers, Helen M.Padua, Maria B.Ware, Stephanie M.2025-04-172025-04-172025Bellchambers HM, Padua MB, Ware SM. A CRISPR mis-insertion in the Zic3 5'UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin. Biol Open. 2025;14(3):bio061677. doi:10.1242/bio.061677https://hdl.handle.net/1805/47097Zic3 loss of function is associated with a range of congenital defects, including heterotaxy and isolated heart defects in humans, as well as neural tube defects, situs anomalies, and tail kinks in model organisms. Here, we describe a novel Zic3ins5V mouse line generated due to a mis-insertion during the CRISPR genome editing process, which altered the Zic3 5'UTR structure. Mice with this insertion developed similar phenotypes to Zic3LacZ null mice, including heterotaxy, isolated heart defects, neural tube defects and tail kinks. Surprisingly, gene expression analysis revealed that the novel Zic3ins5V line displays higher levels of Zic3 mRNA, but western blot analysis confirmed that levels of ZIC3 were greatly reduced in vivo. RNAfold, an RNA secondary structure prediction tool, showed that this mis-insertion may cause the formation of a large stem-loop hairpin incorporating some of the 5'UTR and first exon of Zic3, and the insertion of similar hairpins in a cell-based assay caused the loss of ZIC3 expression. Thus, this mouse line displays a loss of ZIC3 protein consistent with the inhibitory effects of 5'UTR stem-loop hairpin structures.en-USAttribution 4.0 InternationalCRISPRCardiac developmentGene expressionRNA structureTransgenic miceTranslation controlArticle