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

BACKGROUND

Mutations in the CDKL5 gene are strongly associated with severe neurodevelopmental disorders, including atypical Rett syndrome and autism spectrum disorder (ASD).

OBJECTIVES

This study aimed to establish a cell model carrying a targeted CDKL5 mutation using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and to evaluate the phenotypic and molecular consequences.

METHODS

A single-guide RNA (sgRNA) targeting exon 2 of CDKL5 was designed using in silico tools to minimize off-target activity. The sgRNA and Cas9 nuclease were cloned into the pSpCas9(BB)-2A-green fluorescent protein (GFP) (PX458) vector and transfected into human embryonic kidney 293T (HEK293T) cells. Following fluorescence-activated cell sorting (FACS), genome-editing efficiency was quantified using a T7 endonuclease I (T7E1) assay and Sanger sequencing. Homology-directed repair (HDR) mediated by single-stranded oligodeoxynucleotide (ssODN) templates was used to introduce a specific missense mutation (c.172A>T; p.Lys58Met). Edited clones were evaluated for CDKL5 expression using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and cell survival and apoptosis were assessed using Annexin V/propidium iodide (PI) staining and caspase-3 activity assays.

RESULTS

The CRISPR/Cas9 construct achieved a mean on-target editing efficiency of 41.6 ± 3.2%, as determined by T7E1 digestion, and sequencing confirmed a heterozygous insertion in 28% of sorted clones. HDR-mediated precise editing was detected in 12% of clones. RT-qPCR analysis showed a 55% reduction in CDKL5 mRNA levels (P < 0.01) in edited cells, whereas Western blotting demonstrated a corresponding 48% decrease in protein expression. Functionally, mutant cells exhibited 25% lower viability (P < 0.05) and 2.3-fold higher apoptosis rates (P < 0.01) than wild-type controls. No significant off-target mutations were detected at the top five predicted loci.

CONCLUSIONS

We successfully generated a human cell line model of CDKL5-related ASD using CRISPR/Cas9 technology. The mutation induced marked reductions in CDKL5 expression and cell survival. Although this HEK293T-based model provides a useful platform for mechanistic and screening studies, its non-neuronal origin limits its ability to fully recapitulate the neuronal context of CDKL5 deficiency disorder (CDD). This study establishes a novel, precisely engineered human cellular model using advanced CRISPR/Cas9 technology to mechanistically dissect the consequences of CDKL5 deficiency. By introducing a defined missense mutation (c.172A>T; p.Lys58Met) into HEK293T cells, this study provides a robust platform for investigating the role of CDKL5 in neurodevelopmental disorders and supports future therapeutic development.