CRISPR/Cas9 knock‐in toward creating a Rett syndrome cell model with a synonymous mutation in the MECP2 gene

Abstract Background Rett syndrome is an X‐linked dominant neurodevelopmental disease caused by mutation in the methyl‐CpG‐binding protein 2 ( MECP2 ) gene. This gene encodes a methylated DNA‐binding protein, which acts as a transcriptional regulatory factor. The present study aimed to establish a cell model of Rett syndrome with the MECP2 synonymous mutation c.354G>T (p.Gly118Gly). In addition, the molecular mechanism of pathogenesis of this mutation was also investigated. Methods To create a cell line containing the synonymous variant in MECP2 locus, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9‐mediated homology‐directed repair precise gene editing method was used. In addition, employing the synthesis of cDNA, the effect of this variant on splicing was investigated. Results Using this model and molecular analysis, we found that the c.354G>T synonymous variant created a novel 5' cryptic splice donor site within the exon 3 of MECP2 gene, which resulted in the deletion of 25 nucleotides at the 3' end of exon 3 and presumably protein truncation. Conclusions The results of the present study show that an apparently neutral synonymous polymorphism, which may be commonly classified as non‐pathogenic, may indeed lead to the creation of an aberrant splice site, thereby resulting in disease.