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 RNAs direct 2’-O-methylation of rRNA nucleotides. These short RNAs have specific elements in their structure, namely boxes C and D, and also contain 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 RNA 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 from 3 to 5, which was confirmed by RT-PCR and RNA-Seq. The obtained results allowed us to conclude that SNORD75 contains an element for binding of some factors regulating maturation of pre-lncRNA Gas5. We found evidence that METTL3 might be among the factors involved in this process, with Gas5 splicing being m6A-dependent.