Identification of genes involved in cell cycle regulation using arrayed synthetic CRISPR RNA libraries in a multiparameter high-content assay
Poster Apr 18, 2018
Žaklina Strezoska, Matthew R. Perkett, Eldon T. Chou, Elena Maksimova, Emily M. Anderson, Shawn McClelland, Melissa L. Kelley, Annaleen Vermeulen and Anja van Brabant Smith
Here, we used a cell cycle reporter cell line to perform an arrayed, synthetic crRNA:tracrRNA screen targeting 169 genes with four crRNAs per gene with high content analysis (HCA) to identify genes that regulate the cell cycle. Multiple parameters were used to classify cells into different cellular states and phases of the cell cycle: cells with irregularly shaped nuclei, cells in G1 phase, cells either in S or G2 phase, cells in mitosis or with condensed chromatin, and cells with multinuclear DNA component. We used a novel statistical method for hit identification and applied multiple strategies including gene expression analysis, confirmation of genomic insertions and deletions, and validation by orthogonal reagents to identify high confidence target genes with roles in cell cycle regulation. Most hits had multiple positive crRNAs per target gene, enabling identification of target genes with high confidence, demonstrating the power of combining synthetic crRNAs libraries with HCA assays in screening for complex cellular phenotypes in an arrayed format. Given the ease of transfecting RNAs into most cell types, we expect many phenotypic assays to be amenable to arrayed screening with synthetic crRNA libraries.
Multiplexing cell-based assays is possible using 3D culture models that are larger and more complex than monolayers
Real-time detection methods to measure live or dead cells provide much flexibility for multiplexing
All multiplexed assay combinations should be verified using appropriate controls for each 3D cell culture model.