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

Targeting non-coding RNAs with the CRISPR/Cas9 system in human cell lines.

Author information

1
Department of Pharmacology/Toxicology and Cancer Institute, University of Mississippi Medical Center, Jackson, MS 39216, USA.
2
Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001, China.
3
System Biosciences, Mountain View, CA 94043, USA.
4
Department of Pharmacology/Toxicology and Cancer Institute, University of Mississippi Medical Center, Jackson, MS 39216, USA ymo@umc.edu.

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.

PMID:
25414344
PMCID:
PMC4330338
DOI:
10.1093/nar/gku1198
[Indexed for MEDLINE]
Free PMC Article

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