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CRISPR/Cas9-based knockout screening for long non-coding RNA in lung cancer

Author Taisia POLIDORI
Director of thesis Rory Johnson
Co-director of thesis Carlo Carolis
Summary of thesis

CRISPR/Cas9-based knockout screening for long non-coding RNAs in cancer


The human genome contains many thousands of long non-coding RNAs (lncRNAs), the majority of which are still uncharacterised and may play roles in human disease. The aim of the project is to discover lncRNAs that play important roles in cancer, and may be targets for therapy.


We will leverage CRISPR/Cas9 as a tool for discovering lncRNAs with roles in lung cancer. Functional screens will be performed in vitro in human lung cancer cell lines. We will take advantage of two tools that we have developed for knockout of noncoding elements: a vector system called DECKO (Double Excision CRISPR Knockout) and CRISPETa, a bioinformatic pipeline for paired sgRNA design.


n preliminary experiments, we used DECKO deletion vector, together with a quantitative deletion assay, to test CRISPETa designs by deleting an enhancer and exonic fragment of the MALAT1 oncogene.Knockout constructs will be in paired format, in order to delete target gene promoters while minimising disruption to other genomic elements.


We aim to scale up this approach from single-gene studies to complex pooled libraries.


Resulting candidates will be validated individually, and then studied for both their molecular mechanisms and their mutations in patient samples. Finally, we aim to bring selected candidates forward to in vivo studies, which should allow a better understanding functions and roles of lncRNAs in the disease.


In conclusion, the increasing understanding of the mechanisms by which lncRNAs function will allow the identification of the best targets for diagnostics and therapies.

Administrative delay for the defence