The ribonucleoprotein-mediated CRISPR–Cas9 system induces recurrent Aire gene mutations in contrast to the nickase expression vector in murinein vitroorin vivomodels

Abstract

Although in vitro mTEC cultures can help determine the role of the autoimmune regulator (Aire) gene, the mechanisms of Aire mutations were identified using Aire mutant mice. Nevertheless, long-term cultures of mTECs from mice are difficult to establish. To overcome this, we used a CRISPR‒Cas9 system to edit Aire in a murine mTEC line in vitro and mouse embryos. Two ribonucleoprotein (RNP) complexes were designed to separately target Aire exons 6 and 8. NHEJ-derived indels or HDR-derived mutations were obtained. Recurrent NHEJ-derived mutations were observed among editions, one in exon 6 (del 3554G) and the other in exon 8 (del 5676_5677TG), i.e., the exon 6 mutation was kept in an mTEC clone edited in vitro and in vivo in a mouse, and the exon 8 mutation was kept in mTECs in vitro. None of the mutations obtained with the nickase system were recurrent, indicating the participation of the RNP complex.