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Front. Pharmacol. | doi: 10.3389/fphar.2019.01246

Are snoRNAs “CRISPRable”? A report on box C/D snoRNA editing in human cells

 Julia A. Filippova1,  Anastasiya M. Matveeva1, 2,  Evgenii S. Zhuravlev1, Evgenia A. Balakhonova1, Sergey J. Malanin3, Raihan S. Mahmud3, Tatiana V. Grigoryeva3,  Dmitry V. Semenov1, Valentin V. Vlassov1 and  Grigory A. Stepanov1, 2*
  • 1Institute of Chemical Biology and Fundamental Medicine (RAS), Russia
  • 2Novosibirsk State University, Russia
  • 3Institute of Fundamental Medicine and Biology, Kazan Federal University, Russia

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.

Keywords: snoRNA, GAS5, growth-arrest specific transcript 5, Genome editing, CRISPR/Cas9, box C/D RNA, RNA modification, Alternative Splicing, M6A

Received: 07 Jun 2019; Accepted: 27 Sep 2019.

Copyright: © 2019 Filippova, Matveeva, Zhuravlev, Balakhonova, Malanin, Mahmud, Grigoryeva, Semenov, Vlassov and Stepanov. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Dr. Grigory A. Stepanov, Institute of Chemical Biology and Fundamental Medicine (RAS), Novosibirsk, Russia, stepanovga@niboch.nsc.ru