Abstract
Technological advancement has opened the door to systematic genetics in mammalian cells. Genome-scale loss-of-function screens can assay fitness defects induced by partial gene knockdown, using RNA interference, or complete gene knockout, using new CRISPR techniques. These screens can reveal the basic blueprint required for cellular proliferation. Moreover, comparing healthy to cancerous tissue can uncover genes that are essential only in the tumor; these genes are targets for the development of specific anticancer therapies. Unfortunately, progress in this field has been hampered by off-target effects of perturbation reagents and poorly quantified error rates in large-scale screens. To improve the quality of information derived from these screens, and to provide a framework for understanding the capabilities and limitations of CRISPR technology, we derive gold-standard reference sets of essential and nonessential genes, and provide a Bayesian classifier of gene essentiality that outperforms current methods on both RNAi and CRISPR screens. Our results indicate that CRISPR technology is more sensitive than RNAi and that both techniques have nontrivial false discovery rates that can be mitigated by rigorous analytical methods.
Highlights
In the early 1900s, Lucien Cuenot observed unusual patterns of inheritance when studying coat color in mice and, from his many crosses, never produced a single homozygous yellow mouse (Cuenot, 1905; Paigen, 2003)
The Bayesian classifier was developed to evaluate whether the distribution of fold-changes for hairpins targeting a given gene most closely matched the distribution of fold-changes of hairpins targeting training sets of essential genes or nonessential genes using twofold cross-validation to prevent circularity (Fig 1)
We found the median projection onto the first left singular vector (LSV) of its cognate hairpins and measured whether hairpins with median rank or higher rank were enriched in the right tail by a hypergeometric test, yielding 179 genes at a false discovery rate (FDR) of 25% (Benjamini & Hochberg)
Summary
In the early 1900s, Lucien Cuenot observed unusual patterns of inheritance when studying coat color in mice and, from his many crosses, never produced a single homozygous yellow mouse (Cuenot, 1905; Paigen, 2003). Not long after these observations, it was shown that Cuenot’s crosses resulted in what appeared to be non-Mendelian ratios because he had discovered a lethal gene (Castle & Little, 1910). In the past dozen years or so, systematic genomic studies in eukaryotic model systems have defined sets of lethal or essential genes under defined growth conditions, providing a nexus for biologists to study the essential molecular processes that occur during cell growth and proliferation
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