Cloning of human ene in . required the removal of an intragenic Pribnow-Schaller Box before it's Insertion into genomic safe harbor AAVS1 site using CRISPR-Cas9.

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't 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 'safe-harbor' 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.  Therefore, we chose to target the AAVS1 site for the insertion of , 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. : We used the CRISPR Cas9 a genome editing system to insert the gene at AAVS1 site in human cell-lines. We found that human sequence has a "Pribnow- Schaller Box" which allows its expression in bacteria and expression of ABCB11 protein which is toxic to . ; the removal of this was required for successful cloning. We inserted at AAVS1 site in HEK 293T using CRISPR-Cas9 tool.  We also found that the ABCB11 protein has similarity with . endotoxin (lipid A) transporter MsbA. We inserted at AAVS1 site using CRISPR-Cas9; however, the frequency of homologous recombination was very low for this approach to be successful .