{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T05:10:11Z","timestamp":1772169011147,"version":"3.50.1"},"reference-count":36,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2020,12,23]],"date-time":"2020-12-23T00:00:00Z","timestamp":1608681600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001843","name":"Science and Engineering Research Board","doi-asserted-by":"publisher","award":["ECR\/2015\/000275"],"award-info":[{"award-number":["ECR\/2015\/000275"]}],"id":[{"id":"10.13039\/501100001843","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001407","name":"Department of Biotechnology, Ministry of Science and Technology, India","doi-asserted-by":"publisher","award":["BT\/PR15116\/MED\/31\/334\/2016"],"award-info":[{"award-number":["BT\/PR15116\/MED\/31\/334\/2016"]}],"id":[{"id":"10.13039\/501100001407","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"<ns3:p>\n                    <ns3:bold>Background:<\/ns3:bold>\n                    Genomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesn\u2019t cause any foreseeable risk to the host. The AAVS1 site is the genetic location which is disrupted upon integration of adeno associated virus (AAV) and is considered a \u2018safe-harbor\u2019 in human genome because about one-third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man. \u00a0Therefore, we chose to target the AAVS1 site for the insertion of\n                    <ns3:italic>ABCB11<\/ns3:italic>\n                    , a bile acid transporter which is defective in progressive familial intra hepatic cholestasis type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient.\n                  <\/ns3:p>\n                  <ns3:p>\n                    <ns3:bold>Methods<\/ns3:bold>\n                    : We used the CRISPR Cas9 a genome editing system to insert the\n                    <ns3:italic>ABCB11<\/ns3:italic>\n                    gene at AAVS1 site in human cell-lines.\n                  <\/ns3:p>\n                  <ns3:p>\n                    <ns3:bold>Results:<\/ns3:bold>\n                    We found that human\n                    <ns3:italic>ABCB11<\/ns3:italic>\n                    sequence has a \u201cPribnow- Schaller Box\u201d which allows its expression in bacteria and expression of ABCB11 protein which is toxic to\n                    <ns3:italic>E<\/ns3:italic>\n                    .\n                    <ns3:italic>coli<\/ns3:italic>\n                    ; the removal of this was required for successful cloning. We inserted\n                    <ns3:italic>ABCB11<\/ns3:italic>\n                    at AAVS1 site in HEK 293T using CRISPR-Cas9 tool.\u00a0 We also found that the ABCB11 protein has similarity with\n                    <ns3:italic>E<\/ns3:italic>\n                    .\n                    <ns3:italic>coli<\/ns3:italic>\n                    endotoxin (lipid A) transporter MsbA.\n                  <\/ns3:p>\n                  <ns3:p>\n                    <ns3:bold>Conclusions:<\/ns3:bold>\n                    We inserted\n                    <ns3:italic>ABCB11<\/ns3:italic>\n                    at AAVS1 site using CRISPR-Cas9; however, the frequency of homologous recombination was very low for this approach to be successful\n                    <ns3:italic>in vivo<\/ns3:italic>\n                    .\n                  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The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":2,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}