Are Small Nucleolar RNAs “CRISPRable”? A Report on Box C/D Small Nucleolar RNA Editing in Human Cells

Abstract

CRISPR technologies are nowadays widely used for targeted knockout of numerous protein-coding genes and for the study of various processes and metabolic pathways in human cells. Most attention in the genome editing field is now focused on the cleavage of protein-coding genes or genes encoding long non-coding RNAs (lncRNAs), while the studies on targeted knockout of intron-encoded regulatory RNAs are sparse. Small nucleolar RNAs (snoRNAs) present a class of non-coding RNAs encoded within the introns of various host genes and involved in post-transcriptional maturation of ribosomal RNAs (rRNAs) in eukaryotic cells. Box C/D snoRNAs direct 2'-O-methylation of rRNA nucleotides. These short RNAs have specific elements in their structure, namely, boxes C and D, and a target-recognizing region. Here, we present the study devoted to CRISPR/ Cas9-mediated editing of box C/D snoRNA genes in Gas5. We obtained monoclonal cell lines carrying mutations in snoRNA genes and analyzed the levels of the mutant box C/D snoRNA as well as the 2'-O-methylation status of the target rRNA nucleotide in the obtained cells. Mutations in SNORD75 in the obtained monoclonal cell line were shown to result in aberrant splicing of Gas5 with exclusion of exons 3 to 5, which was confirmed by RT-PCR and RNA-Seq. The obtained results suggest that SNORD75 contains an element for binding of some factors regulating maturation of Gas5 pre-lncRNA. We suggest that METTL3/METTL14 is among such factors, and m 6 A-methylation pathways are involved in regulation of Gas5 splicing. Our results shell light on the role of SNORDs in regulating splicing of the host gene.

An Emerging Class of Long Non-coding RNA With Oncogenic Role Arises From the snoRNA Host Genes

“…The induced mutations affected specifically the ability to form secondary Kink-turn structure in each SNORD (SNORD74, SNORD77, and SNORD80). Moreover, the editing of PAM located in the D' box of SNORD75 affected the alternative splicing of SNHG, thus showing that snoRNAs can modulate the alternative splicing of their SNHG of origin by affecting the m6A methyltransferase complex (211). A better alternative to genome editing is the temporal inhibition of SNHGs through siRNAs.…”

Programmable System of Cas13-Mediated RNA Modification and Its Biological and Biomedical Applications

“…METTL3 catalyzes pri-miRNA and promotes miRNA processing with the assistance of the RNA-binding proteins DGCR8 and hnRNPA2/B1 ( Alarcon et al, 2015 ). SNORD75 contains a region for METTL3 recognition, which regulates the splicing event of the host gene GAS5 ( Filippova et al, 2019 ). m 6 A in circRNA is an important regulator of RNA stability that relies on YTHDF2.…”

C/D box snoRNA SNORD113-6/AF357425 plays a dual role in integrin signalling and arterial fibroblast function via pre-mRNA processing and 2′O-ribose methylation

“…Without surviving clones, we cannot prove that the dying clones were indeed knockouts for AF357425, it could simply be that our CRISPR/Cas9 strategy was not successful. However, it has been shown before that other snoRNAs can be knocked out effectively using CRISPR ( 33 ). Furthermore, by looking at OFP expression, we found that the Cas9/sgRNA vector was transfected effectively every time.…”