RISC-y Business: Limitations of Short Hairpin RNA-Mediated Gene Silencing in the Brain and a Discussion of CRISPR/Cas-Based Alternatives

Abstract

Manipulating gene expression within and outside the nervous system is useful for interrogating gene function and developing therapeutic interventions for a variety of diseases. Several approaches exist which enable gene manipulation in preclinical models, and some of these have been approved to treat human diseases. For the last couple of decades, RNA interference (RNAi) has been a leading technique to knockdown (i.e., suppress) specific RNA expression. This has been partly due to the technology's simplicity, which has promoted its adoption throughout biomedical science. However, accumulating evidence indicates that this technology can possess significant shortcomings. This review highlights the overwhelming evidence that RNAi can be prone to off-target effects and is capable of inducing cytotoxicity in some cases. With this in mind, we consider alternative CRISPR/Cas-based approaches, which may be safer and more reliable for gene knockdown. We also discuss the pros and cons of each approach.

Keywords: CRISPR; CRISPRi; Cas13; Cas9; CasRx; miRNA; shRNA; siRNA.

FIGURE 1

(A) General pathway for shRNA and siRNA; from delivery to processing. ShRNA expression cassettes are usually delivered to cells via DNA plasmids or recombinant viruses. Once the shRNA expression cassettes reach the cell nucleus, they can undergo gene transcription. The shRNA, once transcribed, is processed via Dicer and produces a functional siRNA. Synthetic siRNAs can be delivered to cells too. The siRNAs bind to the RISC complex and their target RNA and cleave their target RNA, which renders the RNA inactive. (B) Three graphical representations of common CRISPR technology. From top to bottom. CasRx (Cas13d)/gRNA complex can target specific RNAs for cleavage/destruction. Cas9/gRNA complex can bind to DNA and cleave it. This creates an insertion or deletion after the cell’s DNA repair machinery attempts to fix the DNA double-strand break. dCas9 fused to a transcriptional repression or activation domain (CRISPRi/a) complexed with a gRNA can reduce or enhance transcription of the target gene, respectively.