Z Gastroenterol 2016; 54 - KV362
DOI: 10.1055/s-0036-1587137

Systematic identification of novel lincRNAs involved in chemoresistance using CRISPR/Cas9 screens

T Zhan 1, LI Franke 1, M Arnold 1, M Breinig 1, F Heigwer 1, J Winter 1, MP Ebert 2, M Boutros 1
  • 1Deutsches Krebsforschungszentrum (DKFZ), Abteilung Signalwege und funktionelle Genomik, Heidelberg, Deutschland
  • 2Universitätsmedizin Mannheim (UMM), II. Medizinische Klinik, Mannheim, Deutschland

Einleitung: A large fraction of the human genome consists of non-protein coding genes, including long intergenic non-coding RNAs (lincRNAs). The expression pattern of lincRNAs is specifically altered in different tumor subtypies, indicating an important role of lincRNAs for tumor biology. However, the function of most lincRNAs are poorly characterized so far.

Ziele: Systematic identification of lincRNAs that are involved chemoresistance or synthetic lethality towards 5-fluorouracil, oxaliplatin and SN-38 (active metabolite of irinotecan) treatment in colorectal cancer cells.

Methodik: CRISPR/Cas9 technology was used to functionally deplete lincRNAs. Depletion was achieved by either gene knockout via Cas9 (CRISPRn) or knockdown using CRISPR interference (CRISPRi). Colorectal cancer cell lines stably expressing Cas9 or dCas9-KRAB were infected with a lentiviral sgRNA library targeting 712 lincRNAs with 15 sgRNAs per gene (CRISPRi approach) or targeting 307 lincRNAs with 40 sgRNAs (CRISPRn approach). Pools of knockout cells were treated with 5-fluorouracil, oxaliplatin and SN-38 (active metabolite of irinotecan) and the abundance of individual sgRNAs were determinded by next generation sequencing.

Ergebnis: Screening with CRISPRn identified lincRNAs that are functionally implied in chemoresistance and synthetic lethality in combination with oxaliplatin. Functional depletion of LINC00539 caused resistance towards oxaliplatin, while depletion of CECR7 and MIAT resulted in synthetic lethality. Screening with CRISPRi revealed that LINC009933 is associated with resistance to all three drugs in HCT116, however the phenotype ist potentially caused by overlap with protein coding genes.

Schlussfolgerung: Our pilot screens show that CRISPR screens can readily identify lincRNAs that can modulate cellular response to specific chemotherapeutic drugs in colorectal cancer. Follow-up will be needed to evaluate the phenotypic strength and the molecular mechanisms of the candidate genes.