Long non-coding RNAs (lncRNAs) represent a huge reservoir of potential cancer targets. Such “onco-lncRNAs” have resisted traditional RNAi methods, but CRISPR-Cas9 genome editing now promises functional screens at high throughput and low cost. The unique biology of lncRNAs demands screening strategies distinct from protein-coding genes. The first such screens have identified hundreds of onco-lncRNAs promoting cell proliferation and drug resistance. Ongoing developments will further improve screen performance and translational relevance. Researchers from the University of Bern highlight the potential of CRISPR screening technology for discovering new onco-lncRNAs, and to guide molecular oncologists wishing to apply it to their cancer of interest.
Pooled Screening Workflow of the CRISPR-Del Approach for lncRNAs
(A) Library construction. A pooled oligonucleotide library of pgRNAs is designed, targeting promoters of 1,000 lncRNAs with n = 10 independent constructs. Here the perturbation is CRISPRdel, but the same workflow also applies for CRISPRi/a, which only differ in employing one sgRNA rather than a pair (represented by scissors). The resulting library of 103 unique sequences are synthesized as on oligonucleotide pool, cloned into a vector backbone, and used to produce infectious lentivirus. (B) Stable cell line production. Cells stably expressing Cas9/dCas9 are established by transfection or infection, and selected by antibiotics to remove non-expressing cells. (C) Screening. Stable cells are infected with the lentivirus pool, at a low multiplicity of infction (MOI, 0.3), so that the majority of cells receive one single genomically inserted library sequence. After antibiotic selection to remove uninfected cells, cells are subjected to the phenotypic assay, which separates those displaying phenotype of interest (Pheno+) from the rest (Pheno ). Finally, cell populations are harvested and stored. It is crucial to maintain sufficient coverage of the library at every point in the protocol, so that each library sequence is represented by a large number of individual cells, and no bottlenecks occur that could randomly eliminate certain library sequences. (D) PCR amplification. Genomic DNA is purified, and PCR used to amplify lentivirally inserted sgRNAs. PCR primers contain adaptamers and barcodes, in yellow and blue, respectively, needed for the subsequent NGS sequencing. The PCR product is sequenced and mapped to the library design, in order to count the instances of each library sequence. (E) Sequencing, read alignment and statistical analysis. Three different lncRNAs are shown, each represented by three unique sgRNAs. In the example, NGS reads are represented by colored bars. Examples of neutral (in gray), depleted (in red) and enriched (in green) lncRNAs, are provided.