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

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.