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. 2011 Nov;21(11):1944-54.
doi: 10.1101/gr.122358.111. Epub 2011 Aug 15.

Noncoding RNA gene silencing through genomic integration of RNA destabilizing elements using zinc finger nucleases

Tony Gutschner  1 , Marion Baas, Sven Diederichs
Affiliations

Noncoding RNA gene silencing through genomic integration of RNA destabilizing elements using zinc finger nucleases

Tony Gutschner et al. Genome Res. 2011 Nov.

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.

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Figures

Figure 1.
Figure 1.
Knockdown of the abundant ncRNA MALAT1. (A) The relative expression levels of GAPDH, RPLP0, ACTB, and MALAT1 in A549 cells were determined via qRT–PCR and analyzed using the 2(-ddCt) method (Livak and Schmittgen 2001). RN7SL1 was used as reference gene. Shown is the mean of measurements from two experiments (×10−7) and the standard deviation (SD). (B) Schematic overview of the siRNA and qPCR primer position in the MALAT1 transcript. (C) Targeting of MALAT1 with seven different siRNAs yielded a knockdown to, at maximum, 13% remaining expression. The transcript level was determined via qRT–PCR and analyzed using the 2(-ddCt) method (Livak and Schmittgen 2001) with RN7SL1 as the reference gene (mean + SD; n = 2).
Figure 2.
Figure 2.
Use of RNA destabilizing elements for gene silencing. (A) RDEs that could be used for gene silencing show different silencing mechanisms. AU-rich elements and miRNA-binding sites influence the stability of the whole transcript, whereas poly(A) signals only silence downstream sequences. RNase P substrates and self-cleaving ribozymes can destabilize both upstream and/or downstream sequences, depending on the position and sequences used. (B) Different poly(A) signals were tested for their silencing potency in an in vitro combinatorial approach. A549 cells were transfected with plasmids containing combinations of poly(A) signals. Expression of GFP mRNA and vector-derived RNA was determined via qRT–PCR: Low vector encoded RNA expression indicated a strong silencing efficiency. (C) The poly(A) signals differ in silencing efficiency. The best inhibition of expression downstream from the RDE was observed with the bGH signal (mean of three experiments + SD). (D) The MALAT1-derived mascRNA sequence as RDE. Placing the mascRNA element immediately downstream from the ORF of a protein-coding gene leads to RNase P cleavage. The resulting mRNA upstream of the cleavage site is stabilized by a 5′-m7Cap and a short poly(A)-like moiety contributed by the fragment. The remaining 3′-sequence either gets degraded or is directly processed by RNase Z to yield a pre-mascRNA. The resulting 3′-end of the transcript is very unstable due to the lack of a 5′-m7Cap and a 3′-poly(A) tail, and is rapidly degraded. (E) The mascRNA element was tested for its silencing potency in the same in vitro approach used for the poly(A) signals. The 242-bp fragment was inserted immediately after the GFP ORF in sense or antisense orientation. The mascRNA element in sense orientation silenced downstream sequences as efficiently as a bGH signal (mean of three experiments + SD).
Figure 3.
Figure 3.
Silencing of endogenous genes by integration of RNA destabilizing elements. (A) Genomic MALAT1 region targeted by the ZFNs. The ZFNs cut between the MALAT1 TATA box (blue) and the transcriptional start site (arrow). The binding site for each ZFN is depicted in red. The site-specific integration via HR is mediated through the left and right homology arms surrounding the integration cassette containing GFP and the RDE. (B) Integration constructs used in our MALAT1 gene silencing approach. The constructs were either transfected with or without ZFNs. (C) Overview of the silencing approach. Cells were transfected with a pair of ZFNs and a repair template containing the RDE of choice, e.g., poly(A) signal. The successful gene silencing was validated via qRT–PCR and followed by genotyping. This protocol allowed the creation of single-cell clones with validated genotype and reduction in target gene expression within 6 to 8 wk. (D) Genotype–phenotype relationship of selected clones. Single cell clones (A549 wild-type, CMV-GFP, CMV-GFP-SV40+bGH) were genotyped via integration-sensitive PCR to discriminate between heterozygous and homozygous clones. The effective integration gave rise to a longer PCR product and was not present in wild-type cells. MALAT1 RNA expression was analyzed via RT–PCR using two independent primer pairs, detecting the 5′-end or the 3′-end of MALAT1. Only the homozygous, biallelic integration, including the RDE, yielded an efficient silencing of full-length MALAT1. The detection of RN7SL1 is shown as loading control.
Figure 4.
Figure 4.
Genotype–phenotype correlation in single-cell clones. For 297 clones, the genotype was determined. In a pristine correlation, the lowest MALAT1 levels were only found in homozygous clones harboring biallelic RDE integration (red). The homozygous integration of “CMV-GFP-SV40+bGH” (last column) yielded clones with <0.1% MALAT1 expression (median = 0.14%). Displayed are the MALAT1 expression levels of individual clones, as well as the median expression levels in the individual groups (Cells-to-CT; reference gene: RN7SL1).
Figure 5.
Figure 5.
Gene silencing requires ZFN and homozygous poly(A) signal integration and is more effective than siRNA-mediated knockdown. MALAT1 expression levels were determined by Cells-to-CT qRT–PCR of individual clones with RN7SL1 as standardization control. For each group, average expression levels (±SEM) normalized to MALAT1 expression levels in untreated A549 cells (=100%) are shown. The statistical significance of the detected differences was calculated in t-tests; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (A) Comparison of the same targeting constructs in the presence or absence of the ZFN. Integration of the poly(A) signal was only observed in the presence of the ZFN, and thus, the MALAT1 expression in clones with homozygous integration was more than 100-fold lower than in clones lacking the ZFN. (B) Comparison of heterozygous and homozygous integration of the poly(A) signal. Homozygous integration of the RDE gave rise to significantly more effective gene silencing. (C) Copy number determination of GFP integration sites in 30 clones. Quantitative RT–PCR of genomic DNA from wild-type, heterozygous, and homozygous clones unraveled the number of GFP integration sites relative to sequences upstream of the MALAT1 locus. The number of GFP integration sites matched the copy number of the control sequence in homozygous clones corroborating the specificity of the integration reaction. (D) Comparison of targeting constructs with and without the silencing element, the poly(A) signal(s). While the integration of GFP without an RDE had only a minor effect on MALAT1 expression, homozygous integration of a poly(A) signal significantly reduced MALAT1 expression with one exception. (E) Comparison between RNA interference and the ZFN-mediated RDE integration. Transfection of the four most effective siRNAs was compared with the four clones with strongest poly(A)-induced gene silencing. MALAT1 knockdown was significantly more than 300-fold stronger in the clones with homozygous poly(A) signal integration.
Figure 6.
Figure 6.
Silencing of the protein-coding gene IL2RG with RDE. (A) Genomic IL2RG region on chromosome Xq13.1 targeted by the ZFNs in K562 cells, a female chronic myelogenous leukemia cell line containing an active (Xa) and an inactivated (Xi) X-chromosome. The ZFNs cut in exon 5. The binding site for each zinc finger protein (ZFP) is depicted in red. The site-specific integration via HR is mediated through the left and right homology arms surrounding the integration cassette containing GFP and the RDE (here: bGH or SV40+bGH poly(A) signals). (B) Overview about possible experimental outcomes. Even heterozygous integrations of the RDE can lead to a knockout phenotype if the active X chromosome harbors the integration site. (C) IL2RG expression in individual clones after ZFN-mediated RDE integration. The IL2RG mRNA expression levels were determined by qRT–PCR for 34 wild-type clones of untreated K562 cells as well as for 216 clones with a heterozygous or homozygous RDE integration mediated by a ZFN. Individual clones displayed expression levels around or below 0.1% expression compared with the average IL2RG expression level in untreated K562 clones. (D) Average IL2RG expression after ZFN-mediated RDE integration. On average, the IL2RG mRNA expression was significantly decreased in heterozygous as well as homozygous clones compared with the expression level in wild-type K562 cells. (***) P < 0.001, t-tests.
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