Early and aggressive metastasis is a crucial factor of the dismal prognosis of pancreatic ductaladenocarcinoma (PDAC). Toward identifying processes that are essential to PDAC metastasis, we performed a genome-wide gene knockout screen with a CRISPR-Cas9 library of 259,900 single guide RNA constructs on two sub-clones of a PDAC cell line, which exhibit very low or very high metastatic potential, respectively.

Data comparison identified several genes and pathways that were relevant for metastasising cells. One knockout had a particularly strong effect and was analysed with respect to functional aspects. It was critical for the difference in metastatic potential. Also, a buffering interaction was found between this gene and a master regulator in cancer.

In vivo experiments, namely lung and liver colonisation assays after intravenous and intracardiac cell injections into the bloodstream as well as metastatic behaviour after resection of subcutaneously grown primary tumours, confirmed the very strong effect on metastasis.

Scheme of shRNA knockdown experiments. An shRNA-construct is brought into a cell by means of a lentivirus system. The construct integrates into the genome and the respective shRNA is constitutively expressed at high levels, acting as an inhibitory RNA intracellularly. Using a common primer pair (P1, P2), a barcode sequence that is unique for each shRNA construct can be amplified from each cell.  Therefore, also complex mixtures of cells transduced with different constructs can be studied simultaneously.

RNA interference (RNAi) has become a popular and important tool for the analysis of gene function, although being partly superseeded by CRISPR-Cas already. Loss-of-function studies have greatly facilitated functional analyses of the human transcriptome.
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Limitations of early siRNA experiments, most importantly the transient inhibition of gene expression as well as the inefficient transfection into non-dividing cells, were generally overcome by short hairpin RNA (shRNA) expression vectors, which stably integrate into a target cell's genome via retro- or lentiviral gene transfer. Intracellular processing of shRNAs results in short duplex RNAs with siRNA-like properties. Viral integration ensures not only a broad range of infectable target cell types but also the stable expression of specific shRNAs, resulting in the permanent reduction of the targeted gene product. Complex shRNA expression libraries allow the targeted knockdown of thousands of different genes in a single experiment.
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Using such lentiviral vector shRNA libraries and initially barcode arrays and meanwhile next-generation sequencing analysis for decoding of the pooled RNAi screens, we are able to quantify the abundance of individual shRNAs and thus determine in a complex pool the number of cells infected with an individual shRNA construct.
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We used the technique to predict anti-proliferative effects of individual shRNAs from pooled negative selection screens. By such screens, we identified synthetic-lethal activities toward combination therapies, defined genes which are required for a stem-cell like phenotype and found tumour suppressor genes by in vivo studies. Further studies are under way, both for the elucidation of basic regulative processes associated to cancer and for the identification of pathways that are affected by particular drugs or compounds. In particular, we use the technique for obtaining more detailed information on the functional effects of particularly potentially druggable gene products.