Project description:CRISPR screens are powerful tools to identify key genes that underlie biological processes. One important type of screen uses fluorescence activated cell sorting (FACS) to sort perturbed cells into bins based on the expression level of a marker gene, followed by guide RNA (gRNA) sequencing. Analysis of these data presents multiple statistical challenges due to multiple factors including the discrete nature of the bins and typically small numbers of replicate experiments. To address these challenges, we developed a robust and powerful Bayesian random effects model and software package called Waterbear. Furthermore, we used Waterbear to explore how various experimental design parameters affect statistical power to establish principled guidelines for future screens. Finally, we experimentally validated our model findings that, when using Waterbear for analysis, high power is maintained even at low coverage and a high multiplicity of infection. We anticipate that Waterbear will be of broad utility for analyzing FACS-based CRISPR screens.
Project description:CRISPR screens are powerful tools to identify key genes that underlie biological processes. One important type of screen uses fluorescence activated cell sorting (FACS) to sort perturbed cells into bins based on the expression level of marker genes, followed by guide RNA (gRNA) sequencing. Analysis of these data presents several statistical challenges due to multiple factors including the discrete nature of the bins and typically small numbers of replicate experiments. To address these challenges, we developed a robust and powerful Bayesian random effects model and software package called Waterbear. Furthermore, we used Waterbear to explore how various experimental design parameters affect statistical power to establish principled guidelines for future screens. Finally, we experimentally validated our experimental design model findings that, when using Waterbear for analysis, high power is maintained even at low cell coverage and a high multiplicity of infection. We anticipate that Waterbear will be of broad utility for analyzing FACS-based CRISPR screens.
Project description:Pooled CRISPR screens are a powerful tool to identify regulators of a biological process, but have been limited to phenotypes that affect viability or can be monitored with fluorescent protein reporters. We evaluate two additional strategies for phenotypic enrichment based on quantification of RNA using a Flow-FISH assay or protein phosphorylation through intracellular phospho-staining. These tools will greatly expand the applicability of CRISPR screens since they increase the number of possible molecular phenotypes and assay designs.