Zinc Finger Nuclease-mediated silencing of non-protein-coding RNA

Only ~2% of the human genome encodes protein-coding genes whereas up to 70% of the genome is transcribed into RNA. Only little is known about the functions of the large class of non-protein-coding RNAs (ncRNA). So far, ncRNAs were found to regulate gene expression, pre-mRNA splicing or play a role in chromatin organization.

Insights into gene functions can be obtained by loss-of-function studies, e.g. by RNA interference. However, siRNAs can have off-target effects and some RNAs - especially ncRNAs - are difficult to target due to their nuclear localization, strong secondary structure or high abundance. Recently developed Zinc Finger Nucleases (ZFNs) could overcome the aforementioned limitations. ZFNs are genetically engineered proteins consisting of a sequence-specific DNA-binding domain fused to an endonuclease that creates DNA double-strand breaks. ZFNs can be engineered to target one specific sequence in the genome to allow site-specific modifications. To date, ZFNs are efficiently used for the creation of knockout cells or animals only for protein-coding genes.

Here, we present a method that uses ZFN-mediated integration of RNA destabilizing elements into the human genome. This approach silences, for the first time, a non-protein-coding gene (MALAT1) with the help of these "genomic scissors". MALAT1 expression was successfully decreased more than 1000-fold in a lung cancer cell line, proving this approach over 300-fold more effective than RNAi. In principle, this technique allows the efficient silencing and therefore functional knockout of any gene in the cell, protein-coding or non-protein-coding.