In vivo CRISPR screening for novel noncoding RNA functional targets in glioblastoma models

Abstract

CRISPR (clustered regularly interspaced short palindromic repeat)‐based genetic screens offer unbiased and powerful tools for systematic and specific evaluation of phenotypes associated with specific target genes. CRISPR screens have been utilized heavily in vitro to identify functional coding and noncoding genes in a large number of cell types, including glioblastoma (GB), though no prior study has described the evaluation of CRISPR screening in GB in vivo. Here, we describe a protocol for targeting and transcriptionally repressing GB‐specific long noncoding RNAs (lncRNAs) by CRISPR interference (CRISPRi) system in vivo, with tumor growth in the mouse cerebral cortex. Given the target‐specific parameters of each individual screen, we list general steps involved in transducing guide RNA libraries into GB tumor lines, maintaining sufficient coverage, as well as cortically injecting and subsequently isolating transduced screen tumor cell populations for analysis. Finally, in order to demonstrate the use of this technique to discern an essential lncRNA, HOTAIR, from a nonessential lncRNA, we injected a 1:1 (HOTAIR:control nonessential lncRNA knockdown) mixture of fluorescently tagged U87 GB cells into the cortex of eight mice, evaluating selective depletion of HOTAIR‐tagged cells at 2 weeks of growth. Fluorescently tagged populations were analyzed via flow cytometry for hiBFP (control knockdown) and green fluorescent protein (HOTAIR knockdown), revealing 17% (p = 0.007) decrease in fluorescence associated with HOTAIR knockdown relative to control. The described in vivo CRISPR screening methodology thus appears to be an effective option for identifying noncoding (and coding) genes affecting GB growth within the mouse cortex.

The multifaceted functions of long non-coding RNAHOTAIRin neuropathologies and its potential as a prognostic marker and therapeutic biotarget

“…In a very interesting recently published study, clustered regularly interspaced short palindromic repeat (CRISPR)-based interference was employed to identify the lncRNAs, including HOTAIR which undergo massive expressional changes during the progression of gliomas in vivo, for example in a mouse xenograft model of glioblastoma U87 cells transplanted in the cerebral cortices (Ref. 95). Further, Schisandrin B, a bioactive phytochemical from the Chinese medicinal plant Schisandra chinensis , was demonstrated to harbour significant anti-glioma (apoptotic induction, and reduced cellular viability, migration and invasiveness) potential, at least in part, by repressing the sponging actions of HOTAIR against miR-125a–mTOR (mammalian target of rapamycin) signalling (Ref.…”

Long Non-Coding RNAs: Tools for Understanding and Targeting Cancer Pathways

“…Furthermore, as in the case of protein-coding genes, attempts are being made to perform lncRNA screens in vivo. For example, Attenello et al have described the methodology for CRISPRi screen in vivo for glioblastoma cells and listed an option for screening lncRNA during tumour growth in a mouse model of brain cancer [86]. Liu et al too have used CRSIPRi screen in vivo for investigating the functional roles of Wnt-regulated lncRNAs in pancreatic cancer.…”

(In)Distinctive Role of Long Non-Coding RNAs in Common and Rare Ovarian Cancers

“…Similarly, CRISPR activation (CRISPRa) can be used for sequence-specific activation of gene expression. Besides several large scales, genome-wide deletion of up to several thousand lncRNA loci [291,292], CRISPRi was successfully used for knocking out particular, single lncRNA such as MALAT1 [293], XIST [294], HOTAIR [295], NEAT1 [296] or UCA1 [297]. Furthermore, instead of transcriptional silencing of the whole lncRNA gene, CRISPR/Cas9 could be also used for "repairing" alleles of lncRNAs genes found to be associated with ovarian cancer susceptibility, such as HOTAIR polymorphism rs920778 [298].…”