Combining CRISPR/Cas9 screening with custom engineered reporter cell lines to identify genes required for tubulin formation
Recorded On: 02/06/2018
Phenotypic high-throughput / high content screens have become popular tools for elucidating molecular and genetic pathways in biological systems. Phenomics, or high-dimensional biology, incorporates screening methods that can enable many parameters to be tested in concert under similar or identical conditions, providing a potential wealth of information about a specific biological process. Here we describe the use of a gene-edited reporter cell line, U2OS LMNB1-TUBA1B-ACTB (Sigma-Aldrich CLL1218), to phenotypically detect genes responsible for tubulin formation. CLL1218 was transduced with CAS9 Blasticidin Lentiviral Particles (Sigma-Aldrich LVCAS9BST) and selected. Following selection, the pool was cloned, and derived clones were then screened for CRISPR/Cas9 activity using a known active gRNA. Preferred clones were expanded and banked to be used in a semi-automated high-throughput CRISPR library screens to identify modulators of tubulin expression, formation, and distribution. Proof-of-concept was demonstrated using a set of CRISPR guides specific for vimentin. Creation of a vimentin knock-out in the CLL1218-Cas9 reporter line alters cell morphology that can be visually detected on a variety of imagining platforms, including high-content instruments. This case-study describes an effective methodology to combine multi-pronged gene-editing with phenotypic screening to enrich our knowledge of gene and molecular interactions in complex biological systems. Further, with an expanded array of reporter cell lines at the researcher’s disposal, this type of strategy can be adjusted to dissect many other relevant pathways and phenotypes.
Mark joined Sigma-Aldrich in 2006, and has worked in the areas of biotherapeutic production, stem cell applications and gene regulation. In 2014, Mark was recruited to lead the Cell Design Studio team in the engineering of custom cell lines utilizing ZFN, CRISPR and shRNA technologies. Mark obtained his Ph.D. in Biochemistry and Molecular Biology from Saint Louis University School of Medicine where he used RNAi in Drosophila models to elucidate developmental and biochemical roles for RNA polymerase II-associated transcription factors. Following graduate school, Mark served as a Postdoctoral Fellow at Washington University in St. Louis where he investigated signalling pathways involved in the development of human meningioma.