Generación de líneas isogénicas pluripotentes inducidas de pacientes con Síndrome de Kleefstra

Kleefstra Syndrome (KS), is a rare neurodevelopmental disorder characterized by intellectual disability, autistic features, infantile hypotonia, and distinctive facial characteristics. KS is caused by haploinsufficiency of the euchromatic histone-lysine N-methyltransferase 1 (EHMT1) gene due to heterozygous mutations or deletions, affecting the EHMT1 gene. The EHMT1 gene encodes a histone methyltransferase (HMTase) that transfers methyl groups to lysine 9 of histone H3 (H3K9). This gene is closely related to its paralog, the euchromatic histone-lysine N-methyltransferase 2 (EHMT2) gene, which encodes a HMTase that also methylates H3K9. EHMT1 and EHMT2 can form homo- or heterodimeric complexes in vitro, dependent on their SET domains. This complex mediates the mono- and dimethylation of H3K9, which are marks involved in transcriptional repression. Recent studies have described the presence of a de novo single base substitution in the EHMT2 gene (chr6:31848838C>A NM_006709.5:c.3229G>T) that causes the amino acid change from alanine to serine at position 1077, in a patient with a phenotype overlapping with KS. This study aims to generate a corrected isogenic induced pluripotent stem cells (iPSCs) line using CRISPR/Cas9 techniques. The ND095 iPSC line, derived from this patient's fibroblasts, exhibited altered colony morphology and spontaneous differentiation into neuronal progenitors compared to the control line. Efficient dissociation and viability were achieved using Gentle Cell Dissociation Reagent, generating viable single cells with colony formation capacity. Lipofectamine Stem demonstrated the highest efficiency transfection on green fluorescent protein. The ND095 iPSC line was transfected with the ribonucleoprotein complex using Lipofectamine Stem to correct EHMT2 mutations via CRISPR/Cas9 gene editing. Sanger sequencing of post-sorting and culturing isolated clones revealed no correction efficiency for the c.3229G>T variant. Analysis of transfected iPSC clones and transfected cell pool sequences reveled reduced Cas9 endonuclease activity. In conclusion, this study established a protocol for cell dissociation and transfection of iPSCs. However, no successful correction of EHMT2 through CRISPR/Cas9-mediated genetic editing was observed, likely due to reduced Cas9 activity. Consequently, further research is required to develop more efficient genetic editing protocols.