CRISPRi screening to identify functional long noncoding RNAs in pediatric acute myeloid leukemia

Introduction:

Long noncoding RNAs (lncRNAs) have emerged as developmental stage- and cell context-specific regulators of gene expression, presenting a novel window for targeted therapies in pediatric acute myeloid leukemia (AML).

Methods & Results:

We profiled lncRNA expression in human hematopoietic cells and pediatric AML patient samples, and discovered stem cell and progenitor signatures that are upregulated in AML. To identify functional lncRNAs, 619 genes from 9 distinct signatures were screened in 8 AML cell lines using a high-throughput CRISPRi approach. Essential lncRNA genes identified by MAGeCK were subsequently validated via individual proliferation assays and qPCR (26 hits, 10 validated). Validated lncRNAs situated head-to-head with a coding gene (4 cases) were further examined via complementary CRISPR knockout and RNAi-based experiments, to separate the contribution of each gene. CRISPR-Cas9-based excision, LNA-GapmeR-based silencing, and cellular assays were conducted, in order to further validate and interrogate our top lncRNA candidates.

Conclusion:

Our results directly implicate lncRNA dysregulation in the pathogenesis of pediatric AMLs, and suggest oncogenic lncRNA candidates. Moreover, we highlight the power of multifaceted CRISPR-Cas9 approaches for interrogating complex genomic loci.