Advanced genetic screens and genome engineering
The platform project Z03 provides access to advanced screening methodologies and a versatile toolbox of genetically modified cell lines and organisms for CRC 1324 members to characterize Wnt signaling pathway components. We provide CRISPR-Cas9 and RNAi screening methods in human cell lines, as well as in Drosophila cell lines and a transgenic fly collection for in vivo screening. We can also adapt different screening methodologies upon request. In the last funding period, we successfully used CRISPR-Cas9-mediated genome engineering to generate a diverse set of knockout and endogenously tagged cell lines of Wnt pathway members. For engineering FZD1,2,4,7KO and FZD1,2,4,5,7,8KO multiplex cell lines, we identified specific sgRNAs that target three or five Frizzled (FZD) receptors simultaneously. These cells are nonresponsive to Wnt ligand stimulation and represent an excellent tool to study ligand-receptor interactions. Moreover, we generated knockout cell lines for the Wnt secretion factors Evi and Porcupine, which can be used to study not only the specific effects of Wnt ligands but also to investigate alternative secretory routes of Wnts. In addition to the knockout cell lines, we also engineered endogenously tagged Dishevelled and β-catenin cell lines. We successfully generated a bi-allelic tagged β-catenin HCT116 cell line harbouring simultaneously mClover-tagged wild type and mCherry-tagged mutant β-catenin, which can be used to study wildtype versus mutant β-catenin at the same time. Similarly, we engineered endogenously tagged Dvl2-mEoS cell lines to investigate the role of Dvl in canonical and non-canonical Wnt signaling. To enable screening for new Wnt signaling components in vivo we have optimized a tissue-specific CRISPR-Cas9 mutagenesis system and have produced a large scale transgenic sgRNA library in Drosophila. We have also pioneered the use of CRISPR-Cas12a in Drosophila and showed that it enables efficient multiplex genome editing. In addition, we have generated genome engineered fly lines expressing fluorescently tagged Wnt signaling components from the endogenous locus. In the next funding period, we will continue to provide support for performing high-throughput RNAi and CRISPR-Cas-based screens and for generating knock-out or endogenously tagged cell and fly lines.
- Ambrosi G., O. Voloshanenko, A.F. Eckert, D. Kranz, G.U. Nienhaus, and M. Boutros. 2020. Allele-specific endogenous tagging and quantitative analysis of β-catenin in colon cancer cells. BioRxiv, https://doi.org/10.1101/2020.06.18.159616
- Imkeller, K., G. Ambrosi, M. Boutros, and W. Huber. 2020. gscreend: modelling asymmetric count ratios in CRISPR screens to decrease experiment size and improve phenotype detection. Genome Biol. 21:53. PMID: 32122365
- Lin, Y.C., A. Haas, A. Bufe, S. Parbin, M. Hennecke, O. Voloshanenko, J.C. Gross, M. Boutros, S.P. Acebron, and H. Bastians. 2020. Wnt10b-GSK3β-dependent Wnt/STOP signaling prevents aneuploidy in human somatic cells. Life Science Alliance. 4(1):e202000855. PMID: 33257473
- Port, F., M. Starostecka, and M. Boutros. 2020. Multiplexed conditional genome editing with Cas12a in Drosophila. PNAS 22890:22899. PMID: 32843348
- Port, F., C. Strein, M. Stricker, B. Rauscher, F. Heigwer, J. Zhou, C. Beyersdorffer, J. Frei, A. Hess, K. Kern, L. Lange, N. Langner, R. Malamud, B. Pavlovic, K. Radecke, L. Schmitt, L. Voos, E. Valentini, and M. Boutros. 2020. A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila. eLife. 9. PMID: 32053108
- Wesslowski, J., P. Kozielewicz, X. Wang, H. Cui, H. Schihada, D. Kranz, M.P. Karuna, P. Levkin, J.C. Gross, M. Boutros, G. Schulte, and G. Davidson. 2020. eGFP-tagged Wnt-3a enables functional analysis of Wnt trafficking and signaling and kinetic assessment of Wnt binding to full-length Frizzled. J Biol Chem. 295:8759-8774. PMID: 32381507
- Chaudhary C., S. Hingole, J. Frei, F. Port, D. Strutt, and M. Boutros. 2019. Robust Wnt signaling is maintained by a Wg protein gradient and Fz2 receptor activity in the developing Drosophila wing. Development. 46(15):dev174789.
- Rauscher B., F. Heigwer, L. Henkel, T. Hielscher, and M. Boutros. 2018. Toward an integrated map of genetic interactions in cancer cells. Mol Syst Biol. e7656. PMID: 29467179
- Voloshanenko, O., U. Schwartz, D. Kranz, B. Rauscher, M. Linnebacher, I. Augustin, and M. Boutros. 2018. beta-catenin-independent regulation of Wnt target genes by RoR2 and ATF2/ATF4 in colon cancer cells. Sci Rep. 8:3178. PMID: 29453334
- Voloshanenko, O., P. Gmach, J. Winter, D. Kranz, and M. Boutros. 2017. Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families. FASEB J. 31:4832-4844. PMID: 28733458