Abstract
BackgroundThe introduction of genome-wide shRNA and CRISPR libraries has facilitated cell-based screens to identify loss-of-function mutations associated with a phenotype of interest. Approaches to perform analogous gain-of-function screens are less common, although some reports have utilized arrayed viral expression libraries or the CRISPR activation system. However, a variety of technical and logistical challenges make these approaches difficult for many labs to execute. In addition, genome-wide shRNA or CRISPR libraries typically contain of hundreds of thousands of individual engineered elements, and the associated complexity creates issues with replication and reproducibility for these methods.ResultsHere we describe a simple, reproducible approach using the SB transposon system to perform phenotypic cell-based genetic screens. This approach employs only three plasmids to perform unbiased, whole-genome transposon mutagenesis. We also describe a ligation-mediated PCR method that can be used in conjunction with the included software tools to map raw sequence data, identify candidate genes associated with phenotypes of interest, and predict the impact of recurrent transposon insertions on candidate gene function. Finally, we demonstrate the high reproducibility of our approach by having three individuals perform independent replicates of a mutagenesis screen to identify drivers of vemurafenib resistance in cultured melanoma cells.ConclusionsCollectively, our work establishes a facile, adaptable method that can be performed by labs of any size to perform robust, genome-wide screens to identify genes that influence phenotypes of interest.
Highlights
Forward genetic screens, in which a phenotype of interest is selected from a population of mutagenized individuals, have long been viewed as a powerful tool to uncover novel components of biological systems
Transposon mutagenesis in cultured cells We considered several factors when setting out to develop a cell-based forward mutagenesis screening approach
We generated a piggyBac vector to create a simple, viral-free method to establish stable expression of the SB transposase in mammalian cells by co-transfecting the SB100X vector with a second vector encoding a hyperactive version of the piggyBac transposase [54](Fig. 1)
Summary
In which a phenotype of interest is selected from a population of mutagenized individuals, have long been viewed as a powerful tool to uncover novel components of biological systems. Forward genetic screens have been more challenging to perform in mammalian organisms, in part due to the size and complexity of mammalian genomes. The development of genome-wide shRNA and CRISPR libraries has facilitated cell-based screens to identify loss-of-function mutations associated with specific phenotypes. Hundreds of studies have been reported using either RNAi or CRISPR screens to identify genes associated with a wide variety of phenotypes [38, 42], including extensive work to understand the vulnerabilities of cancer cell lines [28]. The introduction of genome-wide shRNA and CRISPR libraries has facilitated cell-based screens to identify loss-of-function mutations associated with a phenotype of interest. Approaches to perform analogous gainof-function screens are less common, some reports have utilized arrayed viral expression libraries or the CRISPR activation system. Genome-wide shRNA or CRISPR libraries typically contain of hundreds of thousands of individual engineered elements, and the associated complexity creates issues with replication and reproducibility for these methods
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