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- Title
Genome Editing Tools for Functional Analysis of HNF1A and IL6ST Genes.
- Authors
Dobrinić, Paula; Tadić, Vanja; Bočkor, Luka; Markulin, Dora; Tremski, Mihaela; Zoldoš, Vlatka; Vojta, Aleksandar
- 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.
- Subjects
GENOME editing; GENETIC engineering; FUNCTIONAL analysis; GENETIC vectors; LUCIFERASES
- Publication
Croatica Chemica Acta, 2016, Vol 89, Issue 2, p183
- ISSN
0011-1643
- Publication type
Article
- DOI
10.5562/cca2859