CRISPR/Cas9-Engineered HEK293T Cellular Model Harboring the Pathogenic CDKL5 c.172A>T Variant Recapitulates Core...

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Neuroscience

Background:

• CDKL5 gene mutations are linked to severe neurodevelopmental disorders, including atypical Rett syndrome and autism spectrum disorder (ASD).
• Understanding the molecular and phenotypic consequences of CDKL5 mutations is crucial for developing effective treatments.

Purpose of the Study:

• To establish a human cell model with a specific CDKL5 mutation using CRISPR/Cas9 technology.
• To evaluate the impact of the induced mutation on CDKL5 expression, cell viability, and apoptosis.

Main Methods:

• Utilized CRISPR/Cas9 and a single-guide RNA (sgRNA) to target exon 2 of the CDKL5 gene in HEK293T cells.
• Employed homology-directed repair (HDR) to introduce a precise missense mutation (c.172A>T; p.Lys58Met).
• Assessed gene editing efficiency, CDKL5 expression (RT-qPCR, Western blotting), cell viability, and apoptosis (Annexin V/PI staining, caspase-3 assay).

Main Results:

• Achieved significant on-target editing efficiency with CRISPR/Cas9, confirmed by T7E1 assay and sequencing.
• Demonstrated a marked reduction in both CDKL5 mRNA (55%) and protein expression (48%) in edited cells.
• Observed decreased cell viability (25%) and increased apoptosis rates (2.3-fold) in the mutant cell line compared to controls, with no detected off-target mutations.

Conclusions:

• Successfully generated a human cell line model for CDKL5-related ASD using CRISPR/Cas9.
• The engineered mutation significantly impacts CDKL5 expression and cellular functions, providing a platform for mechanistic studies.
• While valuable for research, the non-neuronal origin of HEK293T cells necessitates further studies in neuronal contexts for CDKL5 deficiency disorder (CDD).