{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T07:09:01Z","timestamp":1790665741767,"version":"4.1.0"},"reference-count":6,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2020,5,29]],"date-time":"2020-05-29T00:00:00Z","timestamp":1590710400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/501100006769","name":"Russian Science Foundation","doi-asserted-by":"publisher","award":["16\u201015\u201010128"],"award-info":[{"award-number":["16\u201015\u201010128"]}],"id":[{"id":"10.13039\/501100006769","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["currentprotocols.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["CP Human Genetics"],"published-print":{"date-parts":[[2020,6]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>In neurodegeneration studies, researchers are faced with problems such as limited material availability and late disease manifestation. Cell models provide the opportunity to investigate molecular mechanisms of pathogenesis. Moreover, genome editing technologies enable generation of isogenic cell models of hereditary diseases. Our protocol outlines an approach for introducing an expanded CAG repeat tract into the first exon of the <jats:italic>HTT<\/jats:italic> gene, the Huntington's disease causing mutation. The protocol allows modeling the disease at various severity levels by introducing different numbers of CAG repeats. Furthermore, the protocol can be applicable for modeling other diseases caused by trinucleotide repeat expansion. It is important to note there are many difficulties with cloning repeated sequences and amplification of GC\u2010rich regions. Here, we also propose troubleshooting options, which overcome these problems. The protocol is based on CRISPR\/Cas9\u2010mediated homologous recombination with a uniquely designed donor plasmid harboring an expanded CAG tract flanked with long homology arms. \u00a9 2020 Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Design and assembling donor and CRISPR\/Cas9\u2010expressing plasmids<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Transfection of cells with plasmids and sorting GFP\u2010positive cells<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: PCR screening single\u2010cell clones and validation of the mutant <jats:italic>HTT<\/jats:italic> expression<\/jats:p>","DOI":"10.1002\/cphg.100","type":"journal-article","created":{"date-parts":[[2020,5,29]],"date-time":"2020-05-29T14:16:05Z","timestamp":1590761765000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Introducing an Expanded Trinucleotide Repeat Tract into the Human Genome for Huntington's Disease Modeling <i>In Vitro<\/i>"],"prefix":"10.1002","volume":"106","author":[{"given":"Tuyana","family":"Malankhanova","sequence":"first","affiliation":[{"name":"Federal Research Center Institute of Cytology and Genetics Siberian Branch of the Russian Academy of Sciences  Novosibirsk Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Sorokin","sequence":"additional","affiliation":[{"name":"Federal Research Center Institute of Cytology and Genetics Siberian Branch of the Russian Academy of Sciences  Novosibirsk Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sergey","family":"Medvedev","sequence":"additional","affiliation":[{"name":"Federal Research Center Institute of Cytology and Genetics Siberian Branch of the Russian Academy of Sciences  Novosibirsk Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suren","family":"Zakian","sequence":"additional","affiliation":[{"name":"Federal Research Center Institute of Cytology and Genetics Siberian Branch of the Russian Academy of Sciences  Novosibirsk Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anastasia","family":"Malakhova","sequence":"additional","affiliation":[{"name":"Federal Research Center Institute of Cytology and Genetics Siberian Branch of the Russian Academy of Sciences  Novosibirsk Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2020,5,29]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.stem.2012.04.026"},{"key":"e_1_2_7_3_1","article-title":"Polyglutamine disease modeling: Epitope based screen for homologous recombination using CRISPR\/Cas9 System","volume":"6","author":"An M. C.","year":"2014","journal-title":"PLoS Currents"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2019.02.008"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2013.08.021"},{"key":"e_1_2_7_6_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkt555"},{"key":"e_1_2_7_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/srep05405"}],"container-title":["Current Protocols in Human Genetics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cphg.100","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cphg.100","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cphg.100","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,9,6]],"date-time":"2023-09-06T08:31:28Z","timestamp":1693989088000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cphg.100"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,5,29]]},"references-count":6,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2020,6]]}},"alternative-id":["10.1002\/cphg.100"],"URL":"https:\/\/doi.org\/10.1002\/cphg.100","archive":["Portico"],"relation":{},"ISSN":["1934-8266","1934-8258"],"issn-type":[{"value":"1934-8266","type":"print"},{"value":"1934-8258","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,5,29]]},"assertion":[{"value":"2020-05-29","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e100"}}