Noncoding RNA gene silencing through genomic integration of RNA destabilizing elements using zinc finger nucleases
- 1Helmholtz-University-Group “Molecular RNA Biology & Cancer,” German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280 (B150), 69120 Heidelberg, Germany;
- 2Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 220/221, 69120 Heidelberg, Germany
Abstract
Zinc finger nucleases (ZFNs) allow site-specific manipulation of the genome. So far, the use of ZFNs to create gene knockouts has been restricted to protein-coding genes. However, non-protein-encoding RNAs (ncRNA) play important roles in the cell, although the functions of most ncRNAs are unknown. Here, we describe a ZFN-based method suited for the silencing of protein-coding and noncoding genes. This method relies on the ZFN-mediated integration of RNA destabilizing elements into the human genome, e.g., poly(A) signals functioning as termination elements and destabilizing downstream sequences. The biallelic integration of poly(A) signals into the gene locus of the long ncRNA MALAT1 resulted in a 1000-fold decrease of RNA expression. Thus, this approach is more specific and 300 times more efficient than RNA interference techniques. The opportunity to create a variety of loss-of-function tumor model cell lines in different cancer backgrounds will promote future functional analyses of important long noncoding RNA transcripts.
Footnotes
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↵3 Corresponding author.
E-mail s.diederichs{at}dkfz.de.
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.122358.111.
- Received February 17, 2011.
- Accepted August 10, 2011.
- Copyright © 2011 by Cold Spring Harbor Laboratory Press
