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 <i>Gas5</i>. 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 <i>SNORD75</i> in the obtained monoclonal cell line were shown to result in aberrant splicing of <i>Gas5</i> with exclusion of exons 3 to 5, which was confirmed by RT-PCR and RNA-Seq. The obtained results suggest that <i>SNORD75</i> contains an element for binding of some factors regulating maturation of Gas5 pre-lncRNA. We suggest that METTL3/METTL14 is among such factors, and m⁶A-methylation pathways are involved in regulation of <i>Gas5</i> splicing. Our results shell light on the role of <i>SNORDs</i> in regulating splicing of the host gene.