Non-genetic CRISPR approaches to discover drivers and barriers of neural stem cell identity / The epigenomic landscape of four genes which are important in the context of development and disease
Epithelial to mesenchymal transition (EMT) is a key process during the disease progressions of fibrosis and cancer. Controlling EMT could slow disease progression and help prevent critical steps. To find new factors that can control EMT, I performed two different genome-wide, dCas9-VPR-based CRISPRa screens in a cellular EMT reporter system. One CRISPRa screen was performed with a commercial whole-genome library focusing on the genes' transcription start sites. The other screen was performed with an unfocused whole-genome library produced with the CORALINA method. Potential candidates were identified by existing and custom-made bioinformatic methods to the next-generation sequencing data and verified in validation experiments. According to database and literature searches, all candidates have been described in the context of cancer, but ASPA, LRRC75A, KCTD13, CCNJL, GCN1, ATP5L, VRK3, and ANKAR have not yet been linked directly to EMT regulation. By combining different validated candidates I observed morphological changes that match EMT progression, suggesting a biological potential to impact EMT-related phenotypes.
Publications
Breunig CT, Köferle A, Neuner AM, Wiesbeck MF, Stricker SH (2021) CRISPR tools for physiology and cell state changes: potential of transcriptional engineering and epigenome editing. Physiological Reviews 101.1: 177-211.
Breunig CT, Durovic T, Neuner AM, Baumann V, Wiesbeck MF, Köferle A, Götz M, Ninkovic J, Stricker SH (2018) One step generation of customizable gRNA vectors for multiplex CRISPR approaches through string assembly gRNA cloning (STAgR) PloS one 13.4: e0196015.
Di Giaimo R, Durovic T, Barquin P, Kociaj A, Lepko T, Aschenbroich S, Christopher T Breunig, Martin Irmler, Filippo M Cernilogar, Gunnar Schotta, Joana S Barbosa, Dietrich Trümbach, Emily Violette Baumgart , Andrea M Neuner, Johannes Beckers, Wolfgang Wurst, Stefan H Stricker, Jovica Ninkovic (2018) The aryl hydrocarbon receptor pathway defines the time frame for restorative neurogenesis. Cell Reports 25.12: 3241-3251.
Breunig CT, Neuner AM, Giehrl-Schwab J, Wurst W, Götz M, Stricker SH (2018) A Customizable Protocol for String Assembly gRNA Cloning (STAgR). JoVE (Journal of Visualized Experiments) 142: e58556.
Gerling T, Wagenbauer KF, Neuner AM, Dietz H (2015) Dynamic DNA devices and assemblies formed by shape-complementary, non–base pairing 3D components. Science 347.6229: 1446-1452.