High-Content Screening Approaches That Minimize Confounding Factors in RNAi, CRISPR, and Small Molecule Screening

Screening arrayed libraries of reagents, particularly small molecules began as a vehicle for drug discovery, but the in last few years it has become a cornerstone of biological investigation, joining RNAi and CRISPR as methods for elucidating functional relationships that could not be anticipated, and illustrating the mechanisms behind basic and disease biology, and therapeutic resistance. However, these approaches share some common challenges, especially with respect to specificity or selectivity of the reagents as they are scaled to large protein families or the genome. High-content screening (HCS) has emerged as an important complement to screening, mostly the result of a wide array of specific molecular events, such as protein kinase and transcription factor activation, morphological changes associated with stem cell differentiation or the epithelial-mesenchymal transition of cancer cells. Beyond the range of cellular events that can be screened by HCS, image-based screening introduces new processes for differentiating between specific and nonspecific effects on cells. This chapter introduces these complexities and discusses strategies available in image-based screening that can mitigate the challenges they can bring to screening.