{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,9]],"date-time":"2026-07-09T14:13:13Z","timestamp":1783606393839,"version":"3.55.0"},"posted":{"date-parts":[[2026,7,6]]},"reference-count":24,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,6]],"date-time":"2026-07-06T00:00:00Z","timestamp":1783296000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"name":"NIH","award":["P30CA016087"],"award-info":[{"award-number":["P30CA016087"]}]},{"name":"NIH","award":["RM1HG009491"],"award-info":[{"award-number":["RM1HG009491"]}]},{"name":"NIH","award":["HD028088"],"award-info":[{"award-number":["HD028088"]}]},{"name":"NIH","award":["R01CA265978"],"award-info":[{"award-number":["R01CA265978"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Hirschsprung disease (HSCR) is a complex developmental disorder of the enteric nervous system, primarily driven by regulatory variants within enhancer elements of the\n                  <jats:italic>RET<\/jats:italic>\n                  gene. To investigate how these variants lead to aganglionosis, we developed a humanized mouse model by inserting an intact 77kb human\n                  <jats:italic>RET<\/jats:italic>\n                  genomic locus into the\n                  <jats:italic>Rosa26<\/jats:italic>\n                  safe-harbor locus. Utilizing \u201cbig DNA\u201d synthetic biology and Bxb1-mediated recombination, we integrated the complete human locus including all exons, introns, and upstream regulatory elements which we validated via nanopore and short-read sequencing. Functional analysis confirmed\n                  <jats:italic>in vivo<\/jats:italic>\n                  human\n                  <jats:italic>RET<\/jats:italic>\n                  expression; however, our initial HSCR-associated \u201csensitive\u201d haplotype expressed at only 21% of wild-type levels. This significant reduction proved insufficient to rescue the viability when endogenous mouse\n                  <jats:italic>Ret<\/jats:italic>\n                  was deleted. We identified that this deficiency is partially driven by five risk SNPs within established enhancers. Specifically, using CRISPR\/Cas9 to restore a conserved So\u00d710 binding site (converting a sensitive SNP to a protective one) increased\n                  <jats:italic>RET<\/jats:italic>\n                  expression by 1.9-fold and restored transcription factor binding. This study provides a robust framework for modeling human-specific regulatory disorders and demonstrates the critical impact of non-coding variation on disease pathogenesis.\n                <\/jats:p>","DOI":"10.64898\/2026.07.05.736620","type":"posted-content","created":{"date-parts":[[2026,7,6]],"date-time":"2026-07-06T20:10:20Z","timestamp":1783368620000},"source":"Crossref","is-referenced-by-count":0,"title":["BxbI-mediated insertion of a 77kb human RET sensitive haplotype into the mouse genome to generate a humanized model of Hirschsprung disease"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3165-9110","authenticated-orcid":false,"given":"Ryan D.","family":"Fine","sequence":"first","affiliation":[{"name":"Center for Human Genetics and Genomics, NYU Grossman School of Medicine, New York, NY;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin E.","family":"Low","sequence":"additional","affiliation":[{"name":"Technology Evaluation and Development, The Jackson Laboratory, Bar Harbor, ME;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jarod","family":"Rollins","sequence":"additional","affiliation":[{"name":"Technology Evaluation and Development, The Jackson Laboratory, Bar Harbor, ME;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jon M.","family":"Laurent","sequence":"additional","affiliation":[{"name":"Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Grossman School of Medicine, New York, NY;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael V.","family":"Wiles","sequence":"additional","affiliation":[{"name":"Technology Evaluation and Development, The Jackson Laboratory, Bar Harbor, ME;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aamir","family":"Zuberi","sequence":"additional","affiliation":[{"name":"Technology Evaluation and Development, The Jackson Laboratory, Bar Harbor, ME;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jef D.","family":"Boeke","sequence":"additional","affiliation":[{"name":"Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Grossman School of Medicine, New York, NY;"},{"name":"Department of Biomedical Engineering, NYU Tandon School of Engineering, Brooklyn, NY 11201;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aravinda","family":"Chakravarti","sequence":"additional","affiliation":[{"name":"Center for Human Genetics and Genomics, NYU Grossman School of Medicine, New York, NY;"},{"name":"Department of Neuroscience and Physiology, NYU Grossman School of Medicine, New York, NY"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026070906152187000_2026.07.05.736620v1.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.ge.05.120171.000245"},{"key":"2026070906152187000_2026.07.05.736620v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ajhg.2017.06.005"},{"issue":"2","key":"2026070906152187000_2026.07.05.736620v1.3","first-page":"399","article-title":"The correlation between relatives on the supposition of Mendelian inheritance","volume":"52","year":"1918","journal-title":"Transactions of the Royal Society of Edinburgh"},{"key":"2026070906152187000_2026.07.05.736620v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/s41588-018&ndash;0183-z"},{"key":"2026070906152187000_2026.07.05.736620v1.5","first-page":"568","article-title":"A genetic study of Hirschsprung disease","volume":"46","year":"1990","journal-title":"Am J Hum Genet"},{"key":"2026070906152187000_2026.07.05.736620v1.6","unstructured":"Chakravarti, A. , McCallion, A. 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