Cloning of Human ABCB11 Gene in E. coli required the removal of an Intragenic Pribnow-Schaller Box before its Insertion into Genomic Safe Harbor AAVS1 Site using CRISPR Cas9

Abstract

BackgroundGenomic 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 doesnt cause any foreseeable risk to the host. The AAVS1 site is the site 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 AAVS1 site for the insertion of ABCB11, 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. MethodsWe used CRISPR Cas9 a genome editing tool to insert ABCB11 gene at AAVS1 site in human cell-lines. ResultsWe found that human ABCB11 sequence has a "Pribnow- Schaller Box" which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli and the removal of the same was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that ABCB11 protein has similarity with E. coli Endotoxin (Lipid A) Transporter MsbA. ConclusionWe inserted ABCB11 at AAVS1 site using CRISPR-Cas9, however, the frequency of homologous recombination was very low for this approach to be successful in-vivo (Figure: pictorial abstract). Pictorial Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/284125v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1ae72ddorg.highwire.dtl.DTLVardef@1f72ee8org.highwire.dtl.DTLVardef@128a226org.highwire.dtl.DTLVardef@adacfb_HPS_FORMAT_FIGEXP M_FIG ABCB11 gene (which codes the transporter of human bile salts) is targeted to AAVS1 site using a construct which has 5 and 3 overhangs which are homologous to the AAVS1 site. A Pribnow box was detected inside ABCB11 gene which allowed the gene to transcribe in E. Coli causing bacterial lysis probably through competitive replacement of a homologous transporter protein in E. Coli (E. coli Endotoxin (Lipid A) Transporter) MsbA, resulting in Lipid A (L) accumulation inside the bacteria. C_FIG