Long non-coding RNA tagging and expression manipulation via CRISPR/Cas9-mediated targeted insertion
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Long non-coding RNAs (lncRNAs), defined as RNA transcripts longer than 200 nucleotides without the protein-coding ability (Carninci et al., 2005), share many features with protein-coding messenger RNAs (mRNAs) such as polyadenylated 5′ ends and multi-exonic structures (Guttman et al., 2010). Though expression levels are less abundant, lncRNAs outnumber mRNAs with more diverse regulatory functions (Quinn and Chang, 2016). They may serve as decoys, sponges, signals or scaffolds in regulating chromatin conformation, nuclear organization, gene expression, and protein activity in cis or trans manner (Ulitsky and Bartel, 2013; Quinn and Chang, 2016). LncRNAs are involved in various physiological processes and their loss- or gain-of-function mutations have been implicated in the pathogenesis of human diseases (Wapinski and Chang, 2011). Although their functions have been investigated extensively, manipulation of lncRNAs is challenging, limiting further in-depth analysis for lncRNAs. Efficient and convenient tagging method could be helpful for effective lncRNAs immunoprecipitation to explore lncRNAs-DNA/RNA/protein interactions (Engreitz et al., 2014; Chu et al., 2015). Another challenge is lncRNAs expression manipulation with high efficiency and specificity: Point mutations or insertions and deletions (Indels) are usually insufficient to block lncRNAs functions completely (Cong et al., 2013; Mali et al., 2013). Deleting the whole lncRNA loci or changing lncRNA expression with either clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 system, CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) system have been developed as alternative approaches (Zhu et al., 2016; Liu et al., 2017). However, many lncRNA loci overlap with protein-coding genes and even share common promoter regions, restricting the applications of available tools. In addition, many lncRNAs are cis-acting factors, so traditional overexpression strategy may not work in such conditions. Recently it is shown that targeted insertion could be achieved with CRISPR/Cas9 system via canonical non-homologous end joining (c-NHEJ) pathway without the need for homologous or microhomologous sequences (Schmid-Burgk et al., 2016; Suzuki et al., 2016), so it is plausible to achieve targeted insertion at different sites with one universal donor vector using CRISPR/Cas9 system. As gene trap system has been well-established to disrupt gene functions with selection markers/tags for subsequent functional analysis (Stanford et al., 2001), we here modified gene trap vectors and used CRISPR/Cas9 to establish a scalable tool entitled CTRL (CRISPR-mediated tagging and regulation of lncRNAs) for lncRNA tagging and expression manipulation in mammalian cells. With this method, we successfully tagged lncRNAs at either 5′ or 3′ end. And lncRNA expression status was either stimulated or inhibited reversibly depending on the targeted insertion site.
Targeted insertion inside transcriptional termination site is suitable for lncRNA tagging. However, exogenous fragment insertion inside lncRNA transcripts might disrupt lncRNA functions. Then we designed another six sgRNAs targeting sites after lncRNA transcriptional termination (ZEB1-AS1, PTENP1, DICER1-AS1, TUG1, HOTAIR, and MIAT) and examined the impact of targeted insertions on lncRNA expression in 293T cells (Fig. S3A). Established targeted insertions were confirmed at the genomic level with PCR for all six lncRNAs as described above (Fig. S3B). The expression of all six targeted lncRNAs was also upregulated as described above, though increasing extent was not so dramatic as comparing to cells with targeted insertion inside transcriptional termination site of related lncRNAs (Fig. S3C). The expression changes of neighboring genes were similar as described above, though the upregulation of ZEB1, PTENP1-AS1, and HOXC11 was not so robust as comparing to cells with targeted insertion inside transcriptional termination site of related lncRNAs (Fig. S3D).
As targeted insertion near transcriptional termination sites stimulated lncRNA expression, we wondered whether targeted insertion at transcriptional start site would also change lncRNA expression. Here a modified promoter trap vector containing puromycin-polyA cassette without promoter, a specific sgRNA targeting site and 4× MS2 tagging sequences were designed for targeted insertion at transcriptional start site (Fig. S1C). Puromycin-polyA cassette without promoter would be stimulated by endogenous lncRNA promoter in cells with established targeted insertion, which could be depleted by Cre-mediated recombination (Fig. S4A). Six sgRNAs targeting lncRNA transcriptional start sites (ZEB1-AS1, PTENP1, DICER1-AS1, TUG1, HOTAIR, and MIAT) were designed and their impact on lncRNA expression was examined in 293T cells. Established targeted insertions were examined and confirmed at the genomic level with PCR for all six lncRNAs similarly as described above (Fig. S4B). Then we evaluated the corresponding lncRNA expressions in 293T cells containing established targeted insertion and found that the expression of ZEB1-AS1, PTENP1, HOTAIR, and MIAT was stimulated while DICER1-AS1 and TUG1 were inhibited (Fig. S4C), which indicated complicated regulatory effects for targeted insertion at transcriptional start sites. Expression status of neighboring genes was also examined and it was revealed that the expression of ZEB1, PTENP1-AS1, and HOXC11 was upregulated (Fig. S4D), in consistent with the regulatory relationship observed in cells with established targeted insertion near transcriptional termination sites. Cre-mediated reversion of targeted insertion was also evaluated in 293T cells containing established targeted insertion at transcriptional start sites and expected expression reversion was observed. Upregulated lncRNAs including ZEB1-AS1, PTENP1, HOTAIR, and MIAT were inhibited while downregulated lncRNAs including DICER1-AS1 and TUG1 were induced by Cre recombinase (Fig. S4E). LncRNA-regulated target genes including ZEB1, PTENP1-AS1, and HOXC11 were also reversed by Cre recombinase (Fig. 4F), confirming the regulatory relationship between ZEB1/PTENP1-AS1/HOXC11 and ZEB1-AS1/PTENP1/HOTAIR, respectively.
CRISPR/Cas9-mediated targeted insertion has been used for the tagging and correction of protein-coding RNAs (Schmid-Burgk et al., 2016; Suzuki et al., 2016). Here we combined CRISPR/Cas9 with modified gene trap vectors for lncRNA tagging and expression manipulation. Together with the Cre-LoxP system, we further established conditional targeted insertion system for lncRNA expression manipulation for the first time, which was valuable for lncRNA functional analysis. Recently several lncRNA manipulation tools have been established and applied to a large number of lncRNAs (Zhu et al., 2016; Liu et al., 2017). However, as described above, limitations still exist, making it difficult to elucidate the functions of lncRNAs overlapping with other genes and stimulate lncRNA expression specifically (Quinn and Chang, 2016), which could be overcome by our CTRL system. Taken together, our system provides a valuable tool for comprehensive analysis of lncRNA functions and might be used for high-throughput screening in the future.
We thank Yuefang Zhang and Shifang Shan for their help in experiments. We also thank members of the entire Qiu lab for their help and suggestions. Tian-Lin Cheng is supported by The Knowledge Innovation Program of CAS 2014KIP205, Shanghai Sailing Program 15YF1414200, and NSFC #31600826. Zilong Qiu is supported by NSFC #91432111, #31625013.
Tian-Lin Cheng and Zilong Qiu declares that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors.
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