CRISPR-Based Approaches for the High-Throughput Characterization of Long Non-Coding RNAs

Abstract

Over the last decade, tens of thousands of new long non-coding RNAs (lncRNAs) have been identified in the human genome. Nevertheless, except for a handful of genes, the genetic characteristics and functions of most of these lncRNAs remain elusive; this is partially due to their relatively low expression, high tissue specificity, and low conservation across species. A major limitation for determining the function of lncRNAs was the lack of methodologies suitable for studying these genes. The recent development of CRISPR/Cas9 technology has opened unprecedented opportunities to uncover the genetic and functional characteristics of the non-coding genome via targeted and high-throughput approaches. Specific CRISPR/Cas9-based approaches were developed to target lncRNA loci. Some of these approaches involve modifying the sequence, but others were developed to study lncRNAs by inducing transcriptional and epigenetic changes. The discovery of other programable Cas proteins broaden our possibilities to target RNA molecules with greater precision and accuracy. These approaches allow for the knock-down and characterization of lncRNAs. Here, we review how various CRISPR-based strategies have been used to characterize lncRNAs with important functions in different biological contexts and how these approaches can be further utilized to improve our understanding of the non-coding genome.

3D Chromatin Organization Involving MEIS1 Factor in the cis-Regulatory Landscape of GJB2

“…The CRISPRa complex seems to be less efficient than dCas9-KRAB. The SAM system (CRISPRa) used here is highly efficient at targeting the promoter because it is close to transcriptional machinery, but the target enhancer loses strength [35,36]. To increase activation, we could use an alternative system: enhancer-targeting CRISPRa (enCRISPRa), which seems be more efficient for distal regulatory elements [35].…”

Progress of CRISPR‐based programmable RNA manipulation and detection

“…Cas13 has evolved to target and cleave ssRNA, and it is widely used in diverse cell types (Figure 3a). While Cas9/ Cas12-based knockout and knockdown systems have been applied for functional studies of ncRNAs by targeting DNAs, their limited sensitivity remains a barrier to the effectiveness of these approaches (Hazan & Bester, 2021;Lucere et al, 2020). In contrast, Cas13-based RNA knockdown systems can specifically interfere with RNA transcripts of interest, which could readily attribute observed phonotypes to RNAs.…”

Integration of transcription regulation and functional genomic data reveals lncRNA SNHG6’s role in hematopoietic differentiation and leukemia

“…While these features are important, most do not take into account the cell type-specific functions of lncRNAs. Since most lncRNAs exhibit cell typespecific functionality [1,5], we also included features pertaining to regulation and expression, namely TF binding data from the ENCODE project for lncRNA promoters in four different cell lines for more than 100 TFs [50][51][52]. We then trained the ML algorithm using a set of functionally validated lncRNAs.…”

“…While various perturbation methods have been employed to identify functional lncRNAs, CRISPRi has emerged as the most applied technique across multiple cell types and biological contexts [1].…”