In addition to functional genomics approaches using RNAi, we have begun using CRISPR-Cas9 libraries in primary tumor stem-like cells and other progenitor systems. These studies have included evaluation and prioritization of different types of Cas9 platforms (e.g., Cas9 wt, Cas9n, dCas9-KRAB, Dox-Cas9, Cas9-RNP), evaluation of performance of CRISPR-Cas9 screen analysis methods (e.g., edgeR, MAGeCK, Bayesian classifier), sgRNA prediction algorithms, and creation of custom libraries. Using CRISPR-Cas9 tools, we have published two papers on identification of GBM-lethal genes in brain tumor initiating cells and GBM-specific transcriptional networks (Toledo et al., Cell Reports 2015; Plaisier et al., Cell Systems 2016). More recently, we have created and screened a comprehensive GBM-lethal retest library targeting ~1000 genes, identifying ~50 top GBM-lethal targets. We have also successfully performed genome-wide CRISPR-Cas9 screens in hard-to-screen cell types, including human naïve ESCs and hematopoietic cells. Further, we have created two new types of sgRNA libraries. First is the ProDo (Protein Domain) library that improves upon the current Broad design by preferentially targeting evolutionarily constrained protein domains in genes (with Joshua Meier and Feng Zhang, Broad/MIT). Second is a comprehensive tiling library (cTil), where sgRNA are "tiled" across genes to reveal key gene features that trigger phenotypic penetrance. We have created cTil libraries for the epigenome, kinetochore proteins, and splicing genes.