CRISPR/Cas9 regularly “lights up” our homepage here at Epigenie with new breakthroughs in DNA methylation, stem cell gene editing, and 3-D chromosome structure, to name but a few of the recent highlights. Now, an exciting new study published in Science has employed CRISPR/Cas9 technology to illuminate the “dark matter” of our genome; the ubiquitous but secretive world of the long non-coding RNA (lncRNA).
Our genome contains tens of thousands of lncRNAs, and while these RNAs do not code for proteins, some play critical roles in cellular function, development, and disease. However, thanks to a general lack of specific and scalable tools, the vast majority of lncRNAs still lie in the unknown shadows.
Now, the labs of Jonathan S. Weissman and Daniel A. Lim (University of California, San Francisco, USA) have endeavored to shed light on this genomic dark matter using a CRISPR/Cas9-based technology called CRISPR interference (CRISPRi). CRISPR interference precisely represses gene transcription via the targeted recruitment of the nuclease-dead dCas9-KRAB repressor fusion protein to a transcriptional start site (TSS) with a single guide RNA (sgRNA).
The targeting of a locus leads to the construction of a repressive chromatin environment which blocks the transcription of a targeted gene, or in this case, lncRNA locus, with minimized off-target effects.
In their new study, Liu and Horlbeck et al. employed CRISPR interference to repress 16,000 lncRNA loci (wow!) in various transformed cell lines and human induced pluripotent stem cells (iPSCs) and identified 499 lncRNAs necessary for robust cellular growth.
Such large-scale screening was made possible by the construction of a large sgRNA library, termed “CRiNCL” for CRISPR interference Non-Coding Library, which will be made available to all through the Addgene repository in the near future.
Of note, their results indicated that lncRNA repression disrupted transcriptional networks via distinct molecular mechanisms and, importantly, that a large proportion of lncRNAs (89%) displayed cell-type specificity. The authors note that this situation is unlike that for protein-coding genes, where an essential gene tends to be required for robust growth in a wide range of cell types.
Finally, the study also highlighted the high incidence of cancer-associated single nucleotide polymorphisms (SNPs) in regions containing growth-associated lncRNAs, suggestive of a potentially important link between lncRNAs and cancer development.
This CRISPR-based platform and the availability of the sgRNA library promises to open the floodgates for lncRNA studies and to cast extra light onto this underappreciated, but essential, part of our genome. Future targeted studies will explore the roles of these lncRNAs on higher-order cellular functions and also get to the root of their extraordinary cell-type specificity.
For now, pull up a comfy chair, flick on the reading lamp, and get to know the newly illuminated world of lncRNAs at Science, December 2016.