Comprehensive analysis of hypoxia-regulated long non-coding RNAs in lung adenocarcinoma cells using a single-cell CRISPR-interference-based transcriptional screening - 17/02/23

Current treatments for lung adenocarcinomas (LUAD) have poor response rates and there is an urgent need to propose new strategies to prevent these escape routes. Variability in LUAD drug response is multifactorial including intrinsic altered pathways or extrinsic factors such as hypoxia. Recent advances in cancer genomics have highlighted aberrant expression of ncRNAs as a major determinant of early treatment resistance. We aim here at gaining new insights into the functions of lncRNAs on the hypoxic response of LUAD cells.

We have developed a single-cell CRISPR-interference-based (CRISPRi) transcriptome screening on our best lncRNA candidates based on the CROP-Seq approach (Daltinger et al. Nat Methods 2017). A mini-CROP-seq library including validated gRNAs targeting 8 lncRNA candidates and several key regulators of the hypoxic response (HIF1A, HIF2A) has been amplified and transduced in A549 LUAD cells cultured in normoxia or hypoxia. The cells from the 2 conditions were then pooled, labelled with barcoded antibodies and analyzed by single-cell RNA-Seq in a single run, directly linking guide RNA expression to transcriptome responses in individual cells.

We describe here the precise workflow and the validation of the method on key regulators of the hypoxic response. Moreover, our data indicate that several lncRNAs may be involved in the regulation of cell survival, cell adhesion and/or hypoxic response.

In Conclusion, this method should improve the knowledge on lncRNA-associated functions and provide new potential targets associated with drug resistance in lung cancer.