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

The CRISPR/Cas9 system provides a revolutionary genome editing tool for all areas of molecular biology. In long non-coding RNA (lncRNA) research, the Cas9 nuclease can delete lncRNA genes or introduce RNA-destabilizing elements into their locus. The nuclease-deficient dCas9 mutant retains its RNA-dependent DNA-binding activity and can modulate gene expression when fused to transcriptional repressor or activator domains.

Here, researchers from the German Cancer Research Center systematically analyze whether CRISPR approaches are suitable to target lncRNAs. Many lncRNAs are derived from bidirectional promoters or overlap with promoters or bodies of sense or antisense genes. In a genome-wide analysis, they found only 38% of 15929 lncRNA loci are safely amenable to CRISPR applications while almost two-thirds of lncRNA loci are at risk to inadvertently deregulate neighboring genes. CRISPR- but not siPOOL or Antisense Oligo (ASO)-mediated targeting of lncRNAs NOP14-AS1, LOC389641, MNX1-AS1 or HOTAIR also affects their respective neighboring genes. Frequently overlooked, the same restrictions may apply to mRNAs. For example, the tumor suppressor TP53 and its head-to-head neighbor WRAP53 are jointly affected by the same sgRNAs but not siPOOLs. Hence, despite the advantages of CRISPR/Cas9 to modulate expression bidirectionally and in cis, approaches based on ASOs or siPOOLs may be the better choice to target specifically the transcript from complex loci.

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).