{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T17:49:24Z","timestamp":1790272164605,"version":"4.1.0"},"reference-count":41,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2024,7,30]],"date-time":"2024-07-30T00:00:00Z","timestamp":1722297600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/501100004320","name":"Shiraz University of Medical Sciences","doi-asserted-by":"publisher","award":["21891"],"award-info":[{"award-number":["21891"]}],"id":[{"id":"10.13039\/501100004320","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Cell Biochemistry &amp;amp; Function"],"published-print":{"date-parts":[[2024,8]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:label\/><jats:p>GM1 gangliosidosis is one type of hereditary error of metabolism that occurs due to the absence or reduction of \u03b2\u2010galactosidase enzyme content in the lysosome of cells, including neurons. In vitro, the use of neural cell lines could facilitate the study of this disease. By creating a cell model of GM1 gangliosidosis on the SH\u2010SY5Y human nerve cell line, it is possible to understand the main role of this enzyme in breaking down lipid substrate and other pathophysiologic phenomena this disease. To knock\u2010out the human <jats:italic>GLB1<\/jats:italic> gene, guides targeting exons 14 and 16 of the <jats:italic>GLB1<\/jats:italic> gene were designed using the CRISPOR and CHOP\u2010CHOP websites, and high\u2010efficiency guides were selected for cloning in the PX458 vector. After confirming the cloning, the vectors were transformed into DH5\u03b1 bacteria and then the target vector was extracted and transfected into human nerve cells (SH\u2010SY5Y cell line) by electroporation. After 48\u2009h, GFP<jats:sup>+<\/jats:sup> cells were sorted using the FACS technique and homozygous (compound heterozygous) single cells were isolated using the serial dilution method and sequencing was done to confirm them. Finally, gap PCR tests, X\u2010gal and Periodic acid\u2010Schiff (PAS) staining, and qPCR were used to confirm the knock\u2010out of the human <jats:italic>GLB1<\/jats:italic> gene. Additionally, RNA sequencing data analysis from existing data of the Gene Expression Omnibus (GEO) was used to find the correlation of <jats:italic>GLB1<\/jats:italic> with other genes, and then the top correlated genes were tested for further evaluation of knock\u2010out effects. The nonviral introduction of two guides targeting exons 14 and 16 of the <jats:italic>GLB1<\/jats:italic> gene into SH\u2010SY5Y cells led to the deletion of a large fragment with a size of 4.62\u2009kb. In contrast to the non\u2010transfected cell, X\u2010gal staining resulted in no blue color in <jats:italic>GLB1<\/jats:italic> gene knock\u2010out cells indicating the absence of \u03b2\u2010galactosidase enzyme activity in these cells. Real\u2010time PCR (qPCR) results confirmed the RNA\u2010Seq analysis outcomes on the GEO data set and following the <jats:italic>GLB1<\/jats:italic> gene knock\u2010out, the expression of its downstream genes, <jats:italic>NEU1<\/jats:italic> and <jats:italic>CTSA<\/jats:italic>, has been decreased. It has been also shown that the downregulation of <jats:italic>GLB1<\/jats:italic>\u2010<jats:italic>NEU1<\/jats:italic>\u2010<jats:italic>CTSA<\/jats:italic> complex gene was involved in suppressed proliferation and invasion ability of knock\u2010out cells. This study proved that using dual guide RNA can be used as a simple and efficient tool for targeting the <jats:italic>GLB1<\/jats:italic> gene in nerve cells and the knockout SH\u2010SY5Y cells can be used as a model investigation of basic and therapeutic surveys for GM1 gangliosidosis disease.<\/jats:p><\/jats:sec>","DOI":"10.1002\/cbf.4102","type":"journal-article","created":{"date-parts":[[2024,7,30]],"date-time":"2024-07-30T03:57:11Z","timestamp":1722311831000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Creation of an in vitro model of GM1 gangliosidosis by CRISPR\/Cas9 knocking\u2010out the <i>GLB1<\/i> gene in SH\u2010SY5Y human neuronal cell line"],"prefix":"10.1002","volume":"42","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1189-7143","authenticated-orcid":false,"given":"Kamran","family":"Hosseini","sequence":"first","affiliation":[{"name":"Department of Molecular Medicine, School of Advanced 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