CRISPRi-based radiation modifier screen identifies long non-coding RNA therapeutic targets in glioma

Abstract

Background: Long non-coding RNAs (lncRNAs) exhibit highly cell type-specific expression and function, making this class of transcript attractive for targeted cancer therapy. However, the vast majority of lncRNAs have not been tested as potential therapeutic targets, particularly in the context of currently used cancer treatments. Malignant glioma is rapidly fatal, and ionizing radiation is part of the current standard-of-care used to slow tumor growth in both adult and pediatric patients. Results: We use CRISPR interference (CRISPRi) to screen 5689 lncRNA loci in human glioblastoma (GBM) cells, identifying 467 hits that modify cell growth in the presence of clinically relevant doses of fractionated radiation. Thirty-three of these lncRNA hits sensitize cells to radiation, and based on their expression in adult and pediatric gliomas, nine of these hits are prioritized as lncRNA Glioma Radiation Sensitizers (lncGRS). Knockdown of lncGRS-1, a primate-conserved, nuclear-enriched lncRNA, inhibits the growth and proliferation of primary adult and pediatric glioma cells, but not the viability of normal brain cells. Using human brain organoids comprised of mature neural cell types as a three-dimensional tissue substrate to model the invasive growth of glioma, we find that antisense oligonucleotides targeting lncGRS-1 selectively decrease tumor growth and sensitize glioma cells to radiation therapy. Conclusions: These studies identify lncGRS-1 as a glioma-specific therapeutic target and establish a generalizable approach to rapidly identify novel therapeutic targets in the vast non-coding genome to enhance radiation therapy.

Cell Reprogramming With CRISPR/Cas9 Based Transcriptional Regulation Systems

“…Since the biological functions are established for only a very small proportion of lncRNAs, CRISPR-based screenings can contribute greatly to this field. Although CRISPRn and CRISPRa/i screens have already helped to identify lncRNAs involved in some biological processes ( Liu S. J. et al, 2016 ; Joung et al, 2017 ; Liu et al, 2020 ), there are several issues that make such screening studies complicated. First of all, lncRNAs are distributed throughout the genome, including both the intra- and intergenic regions; moreover, intragenic localization can be intronic, sense, and antisense exonic, or exonicoverlapping with a part of a sense gene.…”

“…lncRNAs can be produced from their own promoters or from promoters shared with other genes ( Awwad, 2019 ). Taken together, these facts show that it could be challenging to apply the CRISPRn screening approach without the risk of disrupting non-target “host” genes or “common” promoters ( Liu et al, 2020 ). Furthermore, small indels, performed by Cas9, do not generally abolish the biological activity since the transcription of lncRNAs, by itself, can have functional consequences ( Kornienko et al, 2013 ).…”

Functional Screening Techniques to Identify Long Non-Coding RNAs as Therapeutic Targets in Cancer

“…CRISPRi is usually achieved by recruiting dCas9-KRAB to the TSS of the target gene. While CRISPRi is able to act within a 1 kb window around the target site, sgRNAs binding within a region of −50 bp upstream to +300 bp downstream of the TSS were found to promote the strongest repression, with maximum activity between +50 bp to +100 bp of the TSS [ 14 , 35 , 45 , 48 ]. CRISPRi screens have successfully identified lncRNAs driving cancer cell growth, as well as lncRNAs contributing to the response of the cell to anti-cancer drugs [ 14 , 45 , 48 ].…”

Identification and characterization of functional long noncoding RNAs in cancer

“…A CRISPR/Cas9‐based genome editing approach used a paired guide RNA (gRNA) strategy to target for deletion a set of 700 human lncRNAs, identifying 51 lncRNAs able to regulate cancer cell growth 116 . Alternatively, CRISPRi (CRISPR inactivation) and CRISPRa (CRISPR activation) screens, involving a nuclease‐dead Cas9 to tether transcriptional repressors or activators to lncRNA loci have provided effective epigenetic loss‐of‐function and gain‐of‐function approaches to query on a genome‐wide level the role of lncRNAs in processes such as cellular proliferation or therapeutic resistance 117‐120 …”