CRISPR-based technology to silence the expression of IncRNAs

James D. Phelan and Louis M. Staudt - PNAS April 14, 2020 117 (15) 8225-8227

The human genome contains more than 3 billion base pairs, and some estimates suggest that nearly 75% of the genome may be transcribed (1), yet only a small fraction (1 to 2%) of the genome that encodes for protein coding regions has been systematically probed for function. The transcribed genome consists of both short noncoding RNAs (ncRNAs) and long ncRNAs (lncRNAs). The lncRNAs are defined as being greater than 200 nucleotides in length and may be capped, polyadenylated, and spliced like protein-coding genes. By definition, lncRNAs do not contain open reading frames of greater than 50 amino acids, but recent work has uncovered a new world of smaller “microproteins” that are encoded by lncRNAs (2). Other functions of lncRNAs include their ability to form aptamers, which bind and regulate protein complexes (3). The lncRNAs can modulate gene expression by enhancer trapping (4), and by recruiting histone modifiers to chromatin (5, 6). These examples notwithstanding, the function of most lncRNAs is unknown. In PNAS, Raffeiner et al. (7) develop CRISPR-based technology to silence the expression of lncRNAs, and use their technology to identify functional lncRNAs that are regulated by the oncogene MYC.

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Inducible CRISPRi screens could reveal phenotypes that are missed in CRISPR screen using catalytically active Cas9. Since gene inactivation by Cas9 is stochastic, the timing of gene inactivation will be variable within a population of sgRNA-expressing cells. By contrast, gene silencing by inducible CRISPRi will be more synchronous, which is a decided advantage when conducting combinatorial genetic screens that aim to knock down two genes at the same time. Further, for some genes, a complete knockout could be cell lethal, but a partial knockdown by CRISPRi could reveal a role in nonlethal phenotypes. Indeed, technologies that partially knock down genes may better model the effect of drugs, which rarely completely inactivate their targets in vivo. And lastly, as demonstrated by Raffeiner et al. (7), CRISPRi technologies are effective tools to probe the noncoding genome. From these perspectives, it will be important to start building a public database of CRISPRi results so that metaanalyses can be performed, which can highlight cell type- and context-specific effects of lncRNAs. The value of such public repositories is evident from the widespread usage of a similar database (Depmap) for Cas9 loss-of-function screens targeting the coding genome (22).

See more: https://www.pnas.org/content/117/15/8225