Targeting Non-Coding RNAs With the CRISPR/Cas9 System in Human Cell Lines

Nucleic Acids Res. 2015 Feb 18;43(3):e17. doi: 10.1093/nar/gku1198. Epub 2014 Nov 20.

Abstract

The CRISPR/Cas has been recently shown to be a powerful genome-editing tool in a variety of organisms. However, these studies are mainly focused on protein-coding genes. The present study aims to determine whether this technology can be applied to non-coding genes. One of the challenges for knockout of non-coding genes is that a small deletion or insertion generated by the standard CRISPR/Cas system may not necessarily lead to functional loss of a given non-coding gene because of lacking an open reading frame, especially in polyploidy human cell lines. To overcome this challenge, we adopt a selection system that allows for marker genes to integrate into the genome through homologous recombination (HR). Moreover, we construct a dual guide RNA vector that can make two cuts simultaneously at designated sites such that a large fragment can be deleted. With these approaches, we are able to successfully generate knockouts for miR-21, miR-29a, lncRNA-21A, UCA1 and AK023948 in various human cell lines. Finally, we show that the HR-mediated targeting efficiency can be further improved by suppression of the non-homologous end joining pathway. Together, these results demonstrate the feasibility of knockout for non-coding genes by the CRISPR/Cas system in human cell lines.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Base Pair Mismatch
  • Blotting, Western
  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Humans
  • MicroRNAs / genetics
  • RNA, Untranslated / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • MicroRNAs
  • RNA, Untranslated