Challenges of CRISPR/Cas9 applications for long non-coding RNA genes

Long non-coding RNAs (lncRNAs) represent a large subgroup of RNAs that are over 200 nucleotides long and have a limited protein-coding potential. They play an important role in diverse cellular processes by regulating the gene expression at transcriptional or post-transcriptional levels. This ability of lncRNAs to modulate gene expression renders them indispensable for normal development

CRISPRs (Clustered Regularly Interspaced Palindromic Repeats) were first discovered in bacteria where they serve as an adaptive immune system against invading phages and plasmid DNA. Given the importance and vast repertoire of uncharacterized lncRNAs, it is tempting to utilize CRISPR-based genomic manipulation tools for their molecular and functional characterization.

Given the complex architecture of genomic loci around lncRNA genes, researchers from the German Cancer Research Center (DKFZ) hypothesize that these intersections can greatly impair the specificity of the potential use of the CRISPR/Cas9 toolbox for the genetic manipulation of lncRNAs due to the possibility of perturbing the overlapping or neighboring genes. This may lead to the identification of phenotypes which in fact would be attributable to neighboring genes. In the present study, the researchers set out to evaluate such risks and examine the utility of different CRISPR/Cas9-based systems for the purposes of lncRNA research.

 Overview of ‘non-CRISPRability’ cases

(A) Top: full-length excision of Intragenic lncRNAs leads to full or partial deletion of the genes they intersect, and therefore cannot be utilized. Bottom: partial deletion of 5΄-proximal genomic regions of lncRNAs arising from non-internal promoters represents a feasible tool for lncRNA manipulation, however bears certain major limitations (see text). (B) CRISPRa/i is not usable for targeting bidirectionally transcribed lncRNAs since the transcription of the adjacent gene might be affected. (C) CRISPRa/i efficiently represses transcription from any promoters located in the vicinity of the desired target site (up to 1.5 – 2 kb), therefore promoters of lncRNAs located in proximity to promoters of other genes cannot be selectively targeted. (D) Top: LncRNAs transcribed from internal promoters cannot be manipulated with CRISPRa/i, as the dCas9 complex binding might affect RNA-Pol II processivity, and thus hinder the transcription of the gene that embeds the internal promoter. Bottom: On the other hand, RNA-Pol II-mediated transcription of the overlapping gene might impair the binding of the dCas9-complex to its target site and therefore reduce the efficiency of CRISPRa/i. CRISPRa or CRISPR activation: dCas9 tagged to a transcriptional activation domain (e.g. VP64); CRISPRi or CRISPR interference: dCas9 alone or dCas9 tagged to a transcriptional inhibitory domain (e.g. KRAB).