Modulation of protein function is used to intervene in cellular processes but is often done indirectly by means of introducing DNA or mRNA encoding the effector protein. Thus far, direct intracellular delivery of proteins has remained challenging. We developed a method termed iTOP, for induced transduction by osmocytosis and propanebetaine, in which a combination of NaCl hypertonicity-induced macropinocytosis and a transduction compound (propanebetaine) induces the highly efficient transduction of proteins into a wide variety of primary cells. We demonstrate that iTOP is a useful tool in systems in which transient cell manipulation drives permanent cellular changes. As an example, we demonstrate that iTOP can mediate the delivery of recombinant Cas9 protein and short guide RNA, driving efficient gene targeting in a non-integrative manner.

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High-content screening (HCS) can generate large multidimensional datasets and when aligned with the appropriate data mining tools, it can yield valuable insights into the mechanism of action of bioactive molecules. However, easy-to-use data mining tools are not widely available, with the result that these datasets are frequently underutilized. Here, we present HC StratoMineR, a web-based tool for high-content data analysis. It is a decision-supportive platform that guides even non-expert users through a high-content data analysis workflow. HC StratoMineR is built by using My Structured Query Language for storage and querying, PHP: Hypertext Preprocessor as the main programming language, and jQuery for additional user interface functionality. R is used for statistical calculations, logic and data visualizations. Furthermore, C++ and graphical processor unit power is diffusely embedded in R by using the rcpp and rpud libraries for operations that are computationally highly intensive. We show that we can use HC StratoMineR for the analysis of multivariate data from a high-content siRNA knock-down screen and a small-molecule screen. It can be used to rapidly filter out undesirable data; to select relevant data; and to perform quality control, data reduction, data exploration, morphological hit picking, and data clustering. Our results demonstrate that HC StratoMineR can be used to functionally categorize HCS hits and, thus, provide valuable information for hit prioritization.