{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T14:51:08Z","timestamp":1786978268800,"version":"build-2736575974"},"reference-count":41,"publisher":"Wiley","issue":"7","license":[{"start":{"date-parts":[[2019,5,23]],"date-time":"2019-05-23T00:00:00Z","timestamp":1558569600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["RA2876\/2-1"],"award-info":[{"award-number":["RA2876\/2-1"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Molec Gen &amp; Gen Med"],"published-print":{"date-parts":[[2019,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>The CRISPR\/Cas9 system has opened new perspectives to study the molecular basis of cerebral cavernous malformations (CCMs) in personalized disease models. However, precise genome editing in endothelial and other hard\u2010to\u2010transfect cells remains challenging.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      In a proof\u2010of\u2010principle study, we first isolated blood outgrowth endothelial cells (BOECs) from a\n                      <jats:italic>CCM1<\/jats:italic>\n                      mutation carrier with multiple CCMs. In a CRISPR\/Cas9 gene correction approach, a high\u2010fidelity Cas9 variant was then transfected into patient\u2010derived BOECs using a ribonucleoprotein complex and a single\u2010strand DNA oligonucleotide. In addition, patient\u2010specific\n                      <jats:italic>CCM1<\/jats:italic>\n                      knockout clones were expanded after CRISPR\/Cas9 gene inactivation.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Deep sequencing demonstrated correction of the mutant allele in nearly 33% of all cells whereas no CRISPR\/Cas9\u2010induced mutations in predicted off\u2010target loci were identified. Corrected BOECs could be cultured in cell mixtures but demonstrated impaired clonal survival. In contrast,\n                      <jats:italic>CCM1<\/jats:italic>\n                      \u2010deficient BOECs displayed increased resistance to stress\u2010induced apoptotic cell death and could be clonally expanded to high passages. When cultured together,\n                      <jats:italic>CCM1<\/jats:italic>\n                      \u2010deficient BOECs largely replaced corrected as well as heterozygous BOECs.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>\n                      We here demonstrate that a non\u2010viral CRISPR\/Cas9 approach can not only be used for gene knockout but also for precise gene correction in hard\u2010to\u2010transfect endothelial cells (ECs). Comparing patient\u2010derived isogenic\n                      <jats:italic>\n                        CCM1\n                        <jats:sup>+\/+<\/jats:sup>\n                      <\/jats:italic>\n                      ,\n                      <jats:italic>\n                        CCM1\n                        <jats:sup>+\/\u2212<\/jats:sup>\n                        ,\n                      <\/jats:italic>\n                      and\n                      <jats:italic>\n                        CCM1\n                        <jats:sup>\u2212\/\u2212<\/jats:sup>\n                      <\/jats:italic>\n                      ECs, we show that the inactivation of the second allele results in clonal evolution of ECs lacking CCM1 which likely reflects the initiation phase of CCM genesis.\n                    <\/jats:p>\n                  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