CRISPR/Cas9-Mediated Regulation of GAS5-Encoded Box-C/D Small Nucleolar RNAs

CRISPR technologies are widely used for a knockout of protein-coding genes or genes encoding long non-coding RNAs (lncRNAs), providing an ability to study and regulate various processes in human cells. However, small non-coding RNAs are a rare target for genome editing. Small nucleolar RNAs (snoRNAs) participate in post-transcriptional modification of ribosomal RNA and guide site-specific 2’-O-methylation of target rRNA nucleotides. Their functioning is possible due to the conservative elements in their structure. Objects of the current study are box-C/D-snoRNAs, encoded in introns of the lncRNA GAS5 (Growth Arrest Specific 5) gene. Location of PAM motifs nearby the conserved structural elements allowed obtaining 293FT-derived single cell clones with mutations in some SNORDs. Cell lines were characterized for mutations in the selected GAS5 regions, target RNA level was estimated as well as the 2’-O-methylation level of corresponding rRNA nucleotides. We were able to obtain lines with the suppressed activity of individual box-C/D small nucleolar RNAs.

Transcriptome of the modified human cell lines was examined using standard RNA-Seq analysis of polyA fraction as well as small RNAs, including small nuclear RNAs, micro-RNAs and snoRNAs. Our findings confirmed the selectivity of CRISPR/Cas9 editing activity regarding snoRNAs of interest. Analysis of the host gene RNA structure demonstrated partial alterations in mature GAS5 RNA, namely, exon skipping and formation of additional non-canonical exon-exon junctions, only for single obtained cell lines.

Thus, the possibility of obtaining viable cell lines with the suppressed activity of a single snoRNA was demonstrated for studying non-canonical functions of individual snoRNAs, as well as their role in gene expression regulation, post-transcriptional maturation of rRNAs and ribosome assembly.

This work was supported by the RFBR grant â„– 18-29-07073.