Klin Padiatr 2016; 228 - A8
DOI: 10.1055/s-0036-1582485

CRISPRi/a screening to identify functional long non-coding RNAs in pediatric acute myeloid leukemia

M Ng 1, L Santer 1, S Emmrich 1, A Schwarzer 2, D Reinhardt 3, JH Klusmann 1
  • 1Pediatric Hematology and Oncology, Hannover Medical School, Hannover
  • 2Hematology, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover
  • 3Paediatric Oncology, University of Duisburg-Essen, Essen, Germany

Introduction: Long non-coding RNAs (lncRNAs) recently emerged as developmental stage and cell context-specific regulators of gene expression, posing a novel window for targeted therapies in pediatric acute myeloid leukemias (AMLs).

Results: We established a microarray-based lncRNA expression atlas for the hematopoietic system including 46 pediatric AML patient samples, and discovered a shared lncRNA signature specific to hematopoietic stem cells (HSCs) and AML blasts. To identify lncRNAs with functions in AML, we first tested CRISPR-Cas9 based transcriptional activator and repressor (CRISPRa/i) systems, and gRNA parameters in fluorescent reporter assays as well as at endogenous loci. CRISPRi was applied for a fluorescence-based drop-out screen in a direct comparison with shRNA-based knockdown. Out of 12 highly expressed candidates from the HSC-AML signature, one lncRNA was found whose knockdown depletes the MLL-rearranged cell line ML-2. Knockdown efficiency was confirmed by qRT-PCR.

Conclusion: Our results represent a comprehensive analysis of the CRISPRi/a systems and demonstrate their capacity for identifying oncogenic lncRNAs, thereby directly implicating lncRNA dysregulation in the pathogenesis of pediatric AML.