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Province","doi-asserted-by":"crossref","award":["2023A1515010176"],"award-info":[{"award-number":["2023A1515010176"]}],"id":[{"id":"10.13039\/501100021171","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["lww.com","ovid.com"],"crossmark-restriction":true},"short-container-title":["JASN"],"published-print":{"date-parts":[[2025,12]]},"abstract":"<jats:sec>\n                    <jats:title>Key Points<\/jats:title>\n                    <jats:p>\n                      <jats:list list-type=\"bullet\">\n                        <jats:list-item>\n                          <jats:p>Magnetic-activated cell sorting\u2013based clustered regularly interspaced short palindromic repeats (CRISPR) screen was used for the first time to identify critical genes and pathways involved in vascular calcification.<\/jats:p>\n                        <\/jats:list-item>\n                        <jats:list-item>\n                          <jats:p>Epigenetic-focused CRISPR screen identified novel vascular calcification regulators, providing potential targets when integrated with transcriptomics.<\/jats:p>\n                        <\/jats:list-item>\n                      <\/jats:list>\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Vascular calcification, mainly driven by osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs), is a common pathologic condition in patients with CKD. However, the roles of other epigenetic regulators in this process remain largely unexplored. Clustered regularly interspaced short palindromic repeats (CRISPR) screen is a highly efficient strategy widely used in identifying genes related to various biologic processes. However, the lack of suitable cell sorting strategies combined with CRISPR screen meant this technology had not been applied to gene screening in vascular calcification.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>We performed an epigenetic-focused CRISPR screen in primary human VSMCs and identified key genes and pathways underlying osteogenic transdifferentiation, based on small guide RNA enrichment in receptor activator of NF-kappa B ligand-positive (calcified) and receptor activator of NF-kappa B ligand-negative (noncalcified) VSMCs isolated by magnetic-activated cell sorting. To validate the screen results, potential genes with different rankings were validated by small interfering RNA intervention and Alizarin Red S staining. Integrating CRISPR results with transcriptome data revealed 17 critical regulators. We further investigated the top hit, anthrax toxin receptor 1 (ANTXR1), in vascular calcification by examining clinical human samples and intervention in mice model.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Through CRISPR screen, we identified 122 potential positive-regulating vascular calcification genes and 116 negative-regulating genes. Phenotypic experimental results further verified the roles of genes with different rankings in osteogenic transdifferentiation of VSMCs, reinforcing the validity of our CRISPR screen system. Notably, integrative analysis of CRISPR screen with transcriptome data revealed ANTXR1 as a critical factor regulating vascular calcification. Furthermore, detection of clinical human samples confirmed the upregulation of ANTXR1 during calcification. Knockdown of\n                      <jats:italic toggle=\"yes\">Antxr1<\/jats:italic>\n                      suppressed vascular calcification in mice model; similarly, overexpression promoted vascular calcification and the osteogenic transdifferentiation of VSMCs.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>Our epigenetic-focused CRISPR screen and transcriptome analysis identified critical epigenetic genes involved in vascular calcification.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1681\/asn.0000000793","type":"journal-article","created":{"date-parts":[[2025,7,18]],"date-time":"2025-07-18T16:00:29Z","timestamp":1752854429000},"page":"2378-2391","update-policy":"https:\/\/doi.org\/10.1097\/lww.0000000000001000","source":"Crossref","is-referenced-by-count":2,"title":["Identification of Epigenetic Regulators of Vascular Calcification with a 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