Generation of Ugt1-Deficient Murine Liver Cell Lines Using TALEN Technology

Abstract

The Crigler-Najjar Syndrome Type I (CNSI) is a rare genetic disorder caused by mutations in the Ugt1a1 gene. It is characterized by unconjugated hyperbilirubinemia that may result in severe neurologic damage and death if untreated. To date, liver transplantation is the only curative treatment. With the aim of generating mutant cell lines of the Ugt1 gene, we utilized the TALEN technology to introduce site-specific mutations in Ugt1 exon 4. We report a fast and efficient method to perform gene knockout in tissue culture cells, based on the use of TALEN pairs targeting restriction enzyme (RE) sites in the region of interest. This strategy overcame the presence of allele-specific single nucleotide polymorphisms (SNPs) and pseudogenes, conditions that limit INDELs' detection by Surveyor. We obtained liver-derived murine N-Muli cell clones having INDELs with efficiency close to 40%, depending on the TALEN pair and RE target site. Sequencing of the target locus and WB analysis of the isolated cell clones showed a high proportion of biallelic mutations in cells treated with the most efficient TALEN pair. Ugt glucuronidation activity was reduced basal levels in the biallelic mutant clones. These mutant liver-derived cell lines could be a very useful tool to study biochemical aspects of Ugt1 enzyme activity in a more natural context, such as substrate specificity, requirement of specific co-factors, the study of inhibitors and other pharmacological aspects, and to correlate enzyme activity to the presence of specific mutations in the gene, by adding back to the mutant cell clones specific variants of the Ugt1 gene. In addition, since genome editing has recently emerged as a potential therapeutic approach to cure genetic diseases, the definition of the most efficient TALEN pair could be an important step towards setting up a platform to perform genome editing in CNSI.

Genome Editing Tools for Functional Analysis of HNF1A and IL6ST Genes

“…[23] In Figure 2, the activity is presented as normalized luminescence. Luminescence measurements were in the range similar to the range reported in the study describing the luciferase assay, [13] which also reported a good correlation between the luciferase assay and in situ targeting. While TALEN-based construct could not induce DSBs above background level at the IL6ST targeted sites, the Cas9-based construct showed activity in a dose-dependent manner.…”

“…[13] To generate the target sequence containing three binding sites for IL6ST-targeting nucleases, 548 bp fragment containing parts of exons 3, 7 and 12 from IL6ST cDNA flanked by BamHI restriction sites was synthetized (Blue Heron Biotech, WA, USA) and cloned into the pGL3-Linker BamHI site. The targeted IL6ST sequences (52 bp long for TALENs and 20 bp long for CRISPR-Cas9) were flanked by about 50 bp of their genomic context on either side, making them spaced about 100 bp relative to each other on the reporter plasmid.…”

“…An elegant assay based on luciferase reporter plasmid has been developed by Porro and coworkers. [13] The luciferase reporter plasmid drives expression of the firefly luciferase gene interrupted by a multiple cloning site (MCS) flanked by two 548 bp repeated regions of luciferase cDNA. The sequence targeted by genome editing tools is cloned into the MCS and upon co-transfection, homologous recombination between flanking repeats reconstitutes luciferase activity in proportion to the frequency of DSBs induced in the cloned targeted region.…”

Liver‐targeted gene therapy: Approaches and challenges

“…Thus, TALENs represent a new and powerful gene editing approach to correct disease‐causing genetic mutations. With this technology, UGT1A1‐deficient mouse liver cell lines were generated to study the CN1 disease, and complete silencing of diacylglycerol acyltransferase‐1 was achieved to abrogate the entry of HCV in Huh‐7.5 cells …”

“…137 Thus, TALENs represent a new and powerful gene editing approach to correct disease-causing genetic mutations. With this technology, UGT1A1-deficient mouse liver cell lines were generated to study the CN1 disease, 160 and complete silencing of diacylglycerol acyltransferase-1 was achieved to abrogate the entry of HCV in Huh-7.5 cells. 161 Clustered Regularly Interspaced Short Palindromic Repeat/Cas9 System The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is the most recent RNA-guided endonuclease technology for genome engineering in mammalian cells (Fig.…”