CRISPRi screening of lncRNAs involved in the Transcriptional Reprogramming of Breast Cancer Cells
LAY SUMMARY
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.
IMPACT
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.
L’Istituto Italiano di Tecnologia (IIT) è una fondazione di diritto privato - cfr. determinazione Corte dei Conti 23/2015 “IIT è una fondazione da inquadrare fra gli organismi di diritto pubblico con la scelta di un modello di organizzazione di diritto privato per rispondere all’esigenza di assicurare procedure più snelle nella selezione non solo nell’ambito nazionale dei collaboratori, scienziati e ricercatori ”.
IIT è sotto la vigilanza del Ministero dell'Istruzione, dell'Università e della Ricerca e del Ministero dell'Economia e delle Finanze ed è stato istituito con la Legge 326/2003. La Fondazione ha l'obiettivo di promuovere l'eccellenza nella ricerca di base e in quella applicata e di favorire lo sviluppo del sistema economico nazionale. La costruzione dei laboratori iniziata nel 2006 si è conclusa nel 2009.
Lo staff complessivo di IIT conta circa 1440 persone. L’area scientifica è rappresentata da circa l’85% del personale. Il 45% dei ricercatori proviene dall’estero: di questi, il 29% è costituito da stranieri provenienti da oltre 50 Paesi e il 16% da italiani rientrati. Oggi il personale scientifico è composto da circa 60 principal investigators, circa 110 ricercatori e tecnologi di staff, circa 350 post doc, circa 500 studenti di dottorato e borsisti, circa 130 tecnici. Oltre 330 posti su 1400 creati su fondi esterni. Età media 34 anni. 41% donne / 59 % uomini.
Nel 2015 IIT ha ricevuto finanziamenti pubblici per circa 96 milioni di euro (80% del budget), conseguendo fondi esterni per 22 milioni di euro (20% budget) provenienti da 18 progetti europei, 17 finanziamenti da istituzioni nazionali e internazionali, circa 60 progetti industriali
La produzione di IIT ad oggi vanta circa 6990 pubblicazioni, oltre 130 finanziamenti Europei e 11 ERC, più di 350 domande di brevetto attive, oltre 12 start up costituite e altrettante in fase di lancio. Dal 2009 l’attività scientifica è stata ulteriormente rafforzata con la creazione di dieci centri di ricerca nel territorio nazionale (a Torino, Milano, Trento, Parma, Roma, Pisa, Napoli, Lecce, Ferrara) e internazionale (MIT ed Harvard negli USA) che, unitamente al Laboratorio Centrale di Genova, sviluppano i programmi di ricerca del piano scientifico 2015-2017.
Istituto Italiano di Tecnologia (IIT) is a public research institute that adopts the organizational model of a private law foundation. IIT is overseen by Ministero dell'Istruzione, dell'Università e della Ricerca and Ministero dell'Economia e delle Finanze (the Italian Ministries of Education, Economy and Finance). The Institute was set up according to Italian law 326/2003 with the objective of promoting excellence in basic and applied research andfostering Italy’s economic development. Construction of the Laboratories started in 2006 and finished in 2009.
IIT has an overall staff of about 1,440 people. The scientific staff covers about 85% of the total. Out of 45% of researchers coming from abroad 29% are foreigners coming from more than 50 countries and 16% are returned Italians. The scientific staff currently consists of approximately 60 Principal Investigators, 110 researchers and technologists, 350 post-docs and 500 PhD students and grant holders and 130 technicians. External funding has allowed the creation of more than 330 positions . The average age is 34 and the gender balance proportion is 41% female against 59% male.
In 2015 IIT received 96 million euros in public funding (accounting for 80% of its budget) and obtained 22 million euros in external funding (accounting for 20% of its budget). External funding comes from 18 European Projects, other 17 national and international competitive projects and approximately 60 industrial projects.
So far IIT accounts for: about 6990 publications, more than 130 European grants and 11 ERC grants, more than 350 patents or patent applications, 12 up start-ups and as many which are about to be launched. The Institute’s scientific activity has been further strengthened since 2009 with the establishment of 11 research nodes throughout Italy (Torino, Milano, Trento, Parma, Roma, Pisa, Napoli, Lecce, Ferrara) and abroad (MIT and Harvard University, USA), which, along with the Genoa-based Central Lab, implement the research programs included in the 2015-2017 Strategic Plan.
