Original scientific paper

https://doi.org/10.5562/cca2859

Genome Editing Tools for Functional Analysis of HNF1A and IL6ST Genes

Paula Dobrinić ; University of Zagreb, Faculty of Science, Department of Biology, Division of Molecular Biology, Horvatovac 102a, HR-10000 Zagreb, Croatia
Vanja Tadić ; University of Zagreb, Faculty of Science, Department of Biology, Division of Molecular Biology, Horvatovac 102a, HR-10000 Zagreb, Croatia
Luka Bočkor ; University of Zagreb, Faculty of Science, Department of Biology, Division of Molecular Biology, Horvatovac 102a, HR-10000 Zagreb, Croatia
Dora Markulin ; University of Zagreb, Faculty of Science, Department of Biology, Division of Molecular Biology, Horvatovac 102a, HR-10000 Zagreb, Croatia
Mihaela Tremski ; University of Zagreb, Faculty of Science, Department of Biology, Division of Molecular Biology, Horvatovac 102a, HR-10000 Zagreb, Croatia
Vlatka Zoldoš ; University of Zagreb, Faculty of Science, Department of Biology, Division of Molecular Biology, Horvatovac 102a, HR-10000 Zagreb, Croatia
Aleksandar Vojta ; University of Zagreb, Faculty of Science, Department of Biology, Division of Molecular Biology, Horvatovac 102a, HR-10000 Zagreb, Croatia

Abstract

Genome editing tools, such as TALEN (transcription activator-like effector nuclease) or CRISPR-Cas9 (CRISPR-associated protein-9 nuclease) systems, enable functional studies by targeted gene knockout. They introduce double-stranded breaks (DSBs) into a DNA molecule in a sequence-specific manner, thereby stimulating the error-prone non-homologous end joining repair mechanism, leading to probable gene inactivation when the coding sequence is targeted. Vectors for expression of TALEN and Cas9-based constructs targeting the human IL6ST and HNF1A genes were assembled and tested for their ability to introduce DSBs when transfected into cultured cells using the luciferase assay. The Cas9-based construct targeting the IL6ST gene was shown to be active, while the two TALEN-based constructs did not introduce DSBs above background level. Both the TALEN and the CRISPR-Cas9 constructs targeting the HNF1A gene were found to be active, with the TALEN showing higher activity in a dose-dependent manner. The constructed genome-editing tools can be used for functional analysis of the putative role of HNF1A and IL6ST genes in IgG glycosylation, as shown previously by genome wide association studies.

This work is licensed under a Creative Commons Attribution 4.0 International License.

Keywords

gene knockout; TALEN; CRISPR; HNF1A; IL6ST; genome editing

Hrčak ID:

161229

URI

https://hrcak.srce.hr/161229

Publication date:

21.6.2016.

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