CRISPRi screening of lncRNAs involved in the Transcriptional Reprogramming of Breast Cancer Cells
Long non-coding RNAs (lncRNAs) are recently discovered regulatory molecules that, unlike messenger RNAs, do not encode proteins. Thousands of them have been discovered so far. Finding out which lncRNAs play a role in cancer has now become a major challenge as they are believed to be extremely promising as diagnostic and therapeutic molecules.
My project consists in using a cutting-edge approach to analyze a multitude of lncRNAs simultaneously and select those that are critical in breast cancer.
My work will provide new insights into the role played by lncRNAs in breast cancer. As lncRNAs are potentially druggable, in the long term our findings might help develop novel therapeutic strategies.
Intro-Background: Long non-coding RNAs (lncRNAs) are long transcripts (>200nt) expressed in a highly specific tissue- and cell-type fashion. They provide a flexible and tunable mechanism of gene expression regulation, acting at transcriptional, post-transcriptional or epigenetic level. Emerging evidence suggests a role for lncRNAs as key components in adaptation pathways. In breast cancer, transcriptional adaptation has been described to occur, in particular during the acquisition of chemoresistance. Unfortunately, the mechanisms behind chemo-adaptation and the role lncRNAs play are still to be clarified. Among the many lncRNAs that exist and are expressed in the breast, we want to find the ones that are crucial for chemo-adaptation, and what are the target genes they control the expression of.
Aim of the project: New technologies for the transcriptional and functional characterization of lncRNAs are emerging. I am using a cutting-edge approach to identify lncRNAs involved in the transcriptional response of TNBC cells that have been exposed to chemotherapy. Specifically, I am performing a CRISPR-based pooled genetic and transcriptional screening to test the ability of candidate lncRNAs to modulate chemo-adaptation. The screening will permit me to identify functional lncRNAs and to characterize their transcriptional targets.
Experimental Design: By characterizing an in vitro TNBC chemo-adaptation model transcriptionally and epigenetically, previous lab members had identified candidate lncRNAs. We designed a library of sgRNAs targeting these lncRNAs and allowing the simultaneous repression of the selected loci by CRISPR-intereference (CRISPRi). I am using a lentiviral library that is barcoded and suitable for single cell RNA sequencing, allowing the dissection of each lncRNA perturbation at both the phenotypic and the transcriptional level.
Expected Output: Our cutting-edge approach will eventually allow the identification of lncRNAs that function in modulating chemo-adaptive responses in triple-negative breast cancer. Moreover, I will be able to dissect the function of these transcripts and unveil the genes they interact with.