CRISPR/Cas9-mediated genome editing in neural progenitor cells

SCK•CEN Mentor

Quintens Roel, rquinten@sckcen.be, +32 (0)14 33 28 37

Expert group

Radiobiology

Introduction

Functional characterization of uncharacterized genes often relies on strategies which ultimately aim to either increase (overexpression) or reduce (knockdown, knockout) the expression of these genes followed by phenotypic analysis. We investigate a newly discovered gene that is transcriptionally activated by p53 in response to radiation-induced DNA damage in mouse embryos and thus may be partly responsible for the observed long-term neurodevelopmental defects. P53-mediated activation of the gene depends on a p53 response element located in an alternative promoter and results in expression of several short transcript variants. Furthermore, this gene is highly upregulated during normal brain development as well as in maturing primary neurons, arguing for a possible role in these important processes.
In this study, we aim to further characterize the function of this gene in neuronal differentiation and maturation via CRISPR/Cas9-mediated gene knockout in mouse neural progenitor cells (NPCs). To highlight the role of this gene in the DNA damage response, we will aim to generate cells in which the p53 response element is mutated, resulting in a cell line in which the gene can no longer be activated by p53. The research project will involve different techniques such as cell culture, CRISPR/Cas9-mediated genome editing, functional assays (e.g. luciferase reporters, live cell imaging), and qRT-PCR.

Objective

Before the start of the project, the student is expected to consult the literature to get acquainted with the project and the molecular techniques that will be used. Also before the start of the project, the student will, together with the supervisor, design appropriate guide RNAs in silico to have them available when the project starts. The ultimate aim will be to have appropriate genomically edited clones for functional validation.

The minimum diploma level of the candidate needs to be

Academic bachelor

The candidate needs to have a background in

Bio-engineering , Biology