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

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

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.

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.

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.

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.