{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T09:26:16Z","timestamp":1783416376122,"version":"3.54.6"},"reference-count":27,"publisher":"Brieflands","issue":"1","license":[{"start":{"date-parts":[[2026,6,8]],"date-time":"2026-06-08T00:00:00Z","timestamp":1780876800000},"content-version":"am","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["brieflands.com"],"crossmark-restriction":false},"short-container-title":["Iran J Pharm Res"],"abstract":"<jats:p>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&gt;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 \u00b1 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 &lt; 0.01) in edited cells, whereas Western blotting demonstrated a corresponding 48% decrease in protein expression. Functionally, mutant cells exhibited 25% lower viability (P &lt; 0.05) and 2.3-fold higher apoptosis rates (P &lt; 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&gt;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.<\/jats:p>","DOI":"10.5812\/ijpr-172311","type":"journal-article","created":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T09:02:22Z","timestamp":1783414942000},"update-policy":"https:\/\/doi.org\/10.5812\/crossmark_update_policy","source":"Crossref","is-referenced-by-count":0,"title":["CRISPR\/Cas9-Engineered HEK293T Cellular Model Harboring the Pathogenic CDKL5 c.172A&amp;gt;T Variant Recapitulates Core Molecular Phenotypes of CDKL5 Deficiency Disorder"],"prefix":"10.5812","volume":"25","author":[{"given":"Fatemeh","family":"Faghihi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"jafar","family":"amani","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Milad","family":"Gholami","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Iman","family":"Salahshourifar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"3819","published-online":{"date-parts":[[2026,6,13]]},"reference":[{"key":"key-AARTICLEREF1-1","doi-asserted-by":"publisher","DOI":"10.1038\/nrg3585"},{"key":"key-AARTICLEREF2-2","doi-asserted-by":"publisher","DOI":"10.1038\/nature11011"},{"key":"key-AARTICLEREF3-3","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1004580"},{"key":"key-AARTICLEREF4-4","doi-asserted-by":"publisher","DOI":"10.1007\/s12035-023-03405-9"},{"key":"key-AARTICLEREF5-5","doi-asserted-by":"publisher","DOI":"10.1038\/nature13908"},{"key":"key-AARTICLEREF6-6","doi-asserted-by":"publisher","DOI":"10.1038\/npjgenmed.2016.27"},{"key":"key-AARTICLEREF7-7","doi-asserted-by":"publisher","DOI":"10.1016\/j.pediatrneurol.2023.04.015"},{"key":"key-AARTICLEREF8-8","doi-asserted-by":"publisher","DOI":"10.1007\/s12031-024-02299-z"},{"key":"key-AARTICLEREF9-9","doi-asserted-by":"publisher","DOI":"10.1155\/2018\/9726950"},{"key":"key-AARTICLEREF10-10","doi-asserted-by":"publisher","DOI":"10.1055\/s-0043-1774528"},{"key":"key-AARTICLEREF11-11","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-12-804066-9.00074-2"},{"key":"key-AARTICLEREF12-12","doi-asserted-by":"publisher","DOI":"10.1186\/s13023-016-0418-y"},{"key":"key-AARTICLEREF13-13","doi-asserted-by":"publisher","DOI":"10.3390\/ijms20205098"},{"key":"key-AARTICLEREF14-14","doi-asserted-by":"publisher","DOI":"10.1038\/s41380-023-01962-y"},{"key":"key-AARTICLEREF15-15","doi-asserted-by":"publisher","DOI":"10.1016\/j.omtn.2025.102634"},{"key":"key-AARTICLEREF16-16","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibtech.2024.07.007"},{"key":"key-AARTICLEREF17-17","doi-asserted-by":"publisher","DOI":"10.1016\/j.bios.2025.117786"},{"key":"key-AARTICLEREF18-18","doi-asserted-by":"publisher","DOI":"10.1016\/j.omtn.2025.102636"},{"issue":"2","key":"key-AARTICLEREF19-19","first-page":"375","volume":"9","author":"Akomodi\u00a0JO","year":"2025","journal-title":"International Journal of Recent Innovations in Academic Research"},{"key":"key-AARTICLEREF20-20","doi-asserted-by":"publisher","DOI":"10.3389\/fnins.2025.1552369"},{"key":"key-AARTICLEREF21-21","doi-asserted-by":"publisher","DOI":"10.1007\/s00431-023-05006-z"},{"key":"key-AARTICLEREF22-22","doi-asserted-by":"publisher","DOI":"10.1007\/s10048-009-0177-1"},{"key":"key-AARTICLEREF23-23","doi-asserted-by":"publisher","DOI":"10.1016\/j.gene.2020.144337"},{"key":"key-AARTICLEREF24-24","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkz1214"},{"key":"key-AARTICLEREF25-25","doi-asserted-by":"publisher","DOI":"10.1007\/s12035-020-01884-8"},{"key":"key-AARTICLEREF26-26","doi-asserted-by":"publisher","DOI":"10.1042\/BST20220791"},{"key":"key-AARTICLEREF27-27","doi-asserted-by":"publisher","DOI":"10.7197\/cmj.1121531"}],"container-title":["Iranian Journal of Pharmaceutical Research"],"original-title":[],"link":[{"URL":"https:\/\/brieflands.com\/journals\/ijpr\/articles\/172311","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/brieflands.com\/journals\/ijpr\/articles\/172311","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T09:02:22Z","timestamp":1783414942000},"score":1,"resource":{"primary":{"URL":"https:\/\/brieflands.com\/journals\/ijpr\/articles\/172311"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,13]]},"references-count":27,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,6,13]]}},"alternative-id":["2276e31ce5f4842dcbc687d31c254e4f18c4b796"],"URL":"https:\/\/doi.org\/10.5812\/ijpr-172311","relation":{},"ISSN":["1726-6890"],"issn-type":[{"value":"1726-6890","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,13]]}}}