Cloning of human ABCB11 gene in E. coli required the removal of an intragenic Pribnow-Schaller Box before it's Insertion into genomic safe harbor AAVS1 site using CRISPR-Cas9.

Nisha Vats,Pankaj Taneja,Madhusudana Girija Sanal,Shiv Kumar Sarin,Senthil Kumar Venugopal

doi:10.12688/f1000research.26659.1

Abstract

Background: 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 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. Methods: We used the CRISPR Cas9 a genome editing system to insert the ABCB11 gene at AAVS1 site in human cell-lines. Results: We 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; the removal of this was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that the ABCB11 protein has similarity with E. coli endotoxin (lipid A) transporter MsbA. Conclusions: We 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.

Highlights

Progressive familial intrahepatic cholestasis type-2 (PFIC2), a severe liver disease which is familial, neonatal, progressive and often fatal which results from a mutation of ATP binding cassette subfamily B member 11 (ABCB11) gene which codes for an ABC transporter bile salt export pump (BSEP)[1,2]
Human ABCB11 gene/product is toxic to E. coli cells Few ampicillin resistant DH5α E. coli colonies which we got after transformation were screened for the insert by colony PCR
We concluded that the ABCB11 gene/gene product is toxic to bacterial cells

Summary

Introduction

Progressive familial intrahepatic cholestasis type-2 (PFIC2), a severe liver disease which is familial, neonatal, progressive and often fatal which results from a mutation of ATP binding cassette subfamily B member 11 (ABCB11) gene which codes for an ABC transporter bile salt export pump (BSEP)[1,2]. In our study we inserted ABCB11 gene at the AAVS1 site using CRISPR-Cas[9] tool (Figure 1) in HEK293T cells and a fibroblast line. We chose to target the AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in progressive familial intra hepatic cholestasis type-2 (PFIC2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient. Methods: We used the CRISPR Cas[9] a genome editing system to insert the ABCB11 gene at AAVS1 site in human cell-lines. Conclusions: We inserted ABCB11 at AAVS1 site using CRISPR-Cas[9]; the frequency of homologous recombination was very low for this approach to be successful in vivo

Methods

Results

Conclusion