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Lennox KA, Behlke MA. J Rare Dis Res Treat. (2016) 1(3): 66-70 Journal of www.rarediseasesjournal.com Rare Diseases Research & Treatment

Mini Review Open Access

Mini-review: Current strategies to knockdown long non-coding Kim A Lennox and Mark A Behlke* Integrated DNA Technologies, Inc., Coralville, IA, 52241, USA

Article Info Text

Article Notes The discovery of functional long non-coding RNAs (lncRNAs) Received: September 12, 2016 has challenged the paradigm that most RNAs encode . Accepted: November 25, 2016 LncRNAs are an abundant class of RNAs >200 nt in length that were *Correspondence: previously assumed to be transcriptional noise. It is now widely Dr. Mark A Behlke, Integrated DNA Technologies, Inc., accepted that some of these lncRNAs have diverse regulatory roles Coralville, IA, 52241, USA, Tel: 319-626-8432; Fax: 319- in expression, can serve as structural components for shaping 626-8466, Email: [email protected] nuclear organization and can even act as decoys for other RNAs © 2016 Mark A Behlke. This article is distributed under the or proteins1-3. While tens of thousands of lncRNA transcripts have terms of the Creative Commons Attribution 4.0 International been cataloged, the biological function of the majority of lncRNAs License. still remains a mystery. Determining the individual functionality Keywords of members of this recently discovered class of RNAs is important Long non-coding RNAs lncRNA for better understanding of developmental biology, evolution and Noncoding RNA genetic disease. Antisense RNAi 4 siRNA distributions that are critical for their function . Some lncRNAs CRISPR are Similarenriched to inproteins, the nucleus individual and are lncRNAs involved have in specific regulating subcellular nuclear Knockdown processes such as and RNA processing. Other lncRNAs are enriched in the where they can impact localization or modulate mRNA stability and . Some lncRNAs are more equally distributed between the nucleus and the cytoplasm. The location of the primary reservoir of a lncRNA can impact the ability to knock down expression levels of that species. Three strategies are commonly used to knockdown or knockout lncRNAs, including: degradation of the RNA by RNA interference (RNAi), degradation of the RNA by RNase H activate antisense (ASOs), or gross deletion/alteration at the DNA level using CRISPR/ genome editing methods (Figure 1). Each of these tools have their own advantages and disadvantages, and

and the transcriptional landscape within which it resides. RNAi is a commonlysuccess can employed be influenced knockdown by the subcellular technique localization that utilizes of the the lncRNA multi- protein RNAi-induced silencing complex (RISC) to suppress mRNAs5. The human RISC loading complex (RLC), is comprised of three proteins (, TRBP and Ago2) responsible for processing longer dsRNAs into the mature siRNAs and loading these siRNAs into Ago2. It has previously been demonstrated that RNAi-mediated mRNA degradation occurs in the cytoplasm6. In a series of fractionation and co-immunoprecipitation experiments, Stalder et al. more

binding with its targeted mRNA and Ago2-mediated cleavage of the mRNAspecifically occurs demonstrate primarily at that the rough canonical endoplasmic RISC loading, reticulum, subsequent where mRNAs are translated into proteins7. RNase H-mediated antisense RNA knockdown capitalizes on the endogenous RNase H1 ,

Page 66 of 70 Lennox KA, Behlke MA. J Rare Dis Res Treat. 2016 1(3): 66-70 Journal of Rare Diseases Research & Treatment

Figure 1: Schematic of the three common methods used to knockdown or knockout lncRNAs. RNA interference (RNAi) utilizes the multi- protein RNAi-induced silencing complex (RISC) containing a siRNA to specifically degrade the targeted RNA. Antisense oligonucleotides (ASOs) bind to their targeted RNA and use endogenous RNase H1, an enzyme that cleaves the RNA in an RNA/DNA heteroduplex. CRISPR-Cas9 genome editing makes alterations at the genomic level by using a target specific crRNA hybridized to the tracrRNA, which is complexed to the Cas9 protein. which is most abundant in the nucleus where it is thought RNase-H mediated antisense knockdown, since RNase to function in DNA replication and repair8-11. Alternatively, H is predominantly found in the nucleus. Conversely, we steric blocking ASOs can be used to block splice junctions thought RNAi, a mostly cytoplasmic process, might be to reduce accumulation of mature lncRNA transcripts or more effective when targeting cytoplasmic populations of block access to key functional domains without triggering lncRNAs. To test this possibility, a systematic study was degradation of the target RNA12,13. Steric blocking ASOs performed in culture comparing the effectiveness of ASOs (20mer DNA-PS, 20mer 2’OMe-PS gapmers or 16mer LNA-PS gapmers (Exiqon), where PS = phosphorothioate morpholinoare made of backbones chemically14. modifiedBoth RNAi residues and RNase that H-active do not linkages, LNA = locked and 2’OMe = 2’-O-Methyl supportASOs rely RNase upon H1 naturally cleavage, present such as effector2’-modified orto RNA) or RNAi (DsiRNAs, siRNA, or Silencer® Select siRNAs degrade the lncRNA. In contrast, CRISPR/Cas9 genome editing methods rely upon a bacterial variable localization patterns17. Nuclear lncRNAs (MALAT1 enzyme that can be targeted to desired sites in the genome (Thermoand NEAT1), Fisher cytoplasmic Scientific)) lncRNAs against (DANCR seven and lncRNAs OIP5-AS1) with or dual-localized lncRNAs (TUG1, CasC7 and HOTAIR) were stranded DNA breaks at or around the target site15,16. targeted by either ASOs (18 sites/target) or RNAi (28 sites/ byThe a cellular site-specific repair guide machinery RNA where heals itthe generates double-stranded double- breaks, leaving small “scars” in the genome, or can even be depending on parameters such as GC content and target used to delete large blocks of DNA and thereby eliminate accessibility,target). As there sites is were a wide selected range by of using efficacy optimized between design sites the lncRNA at the genomic level. CRISPR/Cas9 methods can also be used to introduce new sequences at the target house design criteria (ASOs) to maximize the probability loci, such as transcriptional terminators that will prevent ofalgorithms comparing (RNAi) highly or active using sites well-defined between stringentto the two in- production of full-length lncRNAs. Understanding the knockdown strategies. The results were consistent with nature of the lncRNA of interest can better assist with our hypothesis: nuclear lncRNAs were more easily knocked selecting the optimal knockdown technique. down when using ASOs, while cytoplasmic targets were With an extensive background of mRNA knockdown more easily knocked down when using RNAi (Figure 2). experience, we expected that applying the same repertoire When comparing the potency of the various ASO chemical of reagents to suppress lncRNAs would be easily accomplished. However, we found that some lncRNAs and 2’OMe-PS ASOs had very similar knockdown patterns betweenmodification the strategies,different sites, it was the found 2’OMe-PS that while gapmers the DNA-PS were seemed to be a correlation that RNAi-based methods consistently more potent. This was expected, as the 2’OMe (smallwere difficult interfering to suppress, RNAs, siRNAs; and eventually or Dicer noticed substrate what RNAs, DsiRNAs) were less effective against nuclear- The 2’OMe-PS gapmer and LNA-PS gapmer ASOs typically localized lncRNA targets. We hypothesized that these showedbases increase similar the potency binding at affinity the 10 ofnM the dose; ASO however,to the target. the nuclear lncRNAs would be more easily suppressed using LNA-PS gapmers were often more potent at lower doses.

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Nuclear lncRNAs 120

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Figure 2: Overview of lncRNA knockdown results comparing method efficacy with lncRNA subcellular localization (17). Antisense oligonucleotides (LNA-PS gapmers, 2’OMe-PS gapmers or DNA-PS) or RNAi reagents (siRNAs, DsiRNAs or Silencer® Select siRNAs) were transfected into HeLa cells with ® 2000 for 24 h at 10 nM dose. ASOs = antisense oligonucleotides; PS = phosphorothioate linkages.

There was no overall difference in activity between any of So far, we have commented on the “relative ease” of the various RNAi reagents tested. Duration of knockdown either knockdown technique to suppress their targets was not addressed in this study; suppression lasting 3-7 based on whether the lncRNA was predominantly nuclear days is typically seen in (where dilution from or cytoplasmic. A closer look at the data shows that ASOs cell division can be limiting by reducing concentration are also effective at degrading cytoplasmic RNAs (Figure of the ASOs or siRNAs), whereas knockdown for several 2). ASOs not only have the innate ability to target nuclear- weeks or even a month can be seen in non-dividing retained RNAs, but they can also degrade nascent RNAs cells in vivo18,19. While this study only examined lncRNA prior to cytoplasmic exportation. Also, a recent study knockdown in cell culture, similar relative activities examining the rate-limiting step of mRNA degradation between the different reagent classes would be expected by ASOs found that RNase H1 was also present in low in vivo if suitable delivery tools were employed. For in vivo levels in the cytoplasm23. While ASO-mediated mRNA use, a clear performance advantage would be predicted degradation occurred more rapidly in the nucleus, it also for the short LNA-PS gapmers if administered by naked IV occurred at a slower rate in the cytoplasm. This suggests injection (without any delivery aid)20. Use of a delivery tool, that if the localization of the lncRNA is unknown, selecting such as a (LNP) or conjugated ligand ASO knockdown (as opposed to RNAi) would have the (such as cholesterol or an antibody) can improve delivery best chance of success. Interestingly, our study showed of ASOs and would be required for use of any RNAi reagents that when targeting dual-localized lncRNAs, improved in vivo18,19,21,22. knockdown could be achieved by combining ASOs and

Page 68 of 70 Lennox KA, Behlke MA. J Rare Dis Res Treat. 2016 1(3): 66-70 Journal of Rare Diseases Research & Treatment siRNAs together. This was predictable, considering that interpretation. In fact, Goyal et al recently demonstrated the ASO could most rapidly degrade the nuclear lncRNA that 62% of the 15,929 lncRNAs analyzed in their study population and the siRNA could most rapidly degrade the were poor targets for CRISPR/Cas9 genome editing due to cytoplasmic lncRNA population. containing bidirectional or internal promoters; thus, their Some lncRNAs will be refractory to knockdown by knockout could potentially affect neighboring . The either ASOs or RNAi. This could occur if the subcellular authors selected a few examples from this analysis, and localization of the lncRNA is not accessible to either RNase demonstrated that using CRISPRi to knockout lncRNAs H or the RNAi machinery. It could also occur if the lncRNA with loci that overlap other genes did indeed affect their is highly structured or blocked due to excessive protein neighboring genes. In contrast, RNAi or ASO knockdown binding or hybridizing to other cellular nucleic acids. We 28 tested a wide variety of ASOs and RNAi reagents against transcript . of these same lncRNA targets was specific to the targeted NRON, a lncRNA that functions as a protein sponge, All techniques that suppress have a risk and were unable to achieve high levels of knockdown of off-target effects (OTEs), where related genes that have (unpublished results). In these circumstances, using a sequence homology to the intended target are suppressed technique such as CRISPR/Cas9 genome editing might by cross-hybridization with the targeting nucleic acids. be necessary. Using a genome editing tool to knockout a lncRNA is not trivial, as making a small indel in the targeted nucleic acid hybridization and OTEs are easier to predict lncRNA most likely will not disrupt transcription; no ‘open byThe bioinformatic specificity of ASOsanalysis relies than solely other upon methods; Watson-Crick these reading frame’ exists that is easily disrupted by out-of- frame indel events. Transcribed lncRNAs containing newly 29. While RNAi methods can introduced indels may still retain functional domains and/ reagents can show single-base specificity, especially when or binding sites that would obscure phenotypic analysis. A riskusing of high unsuspected affinity short OTEs ASOs due to crosstalk with miRNA couple of strategies have successfully employed CRISPR/ show single-base specificity, these methods have a higher Cas9 genome editing to knockout lncRNAs. One strategy uses dual guide RNAs to simultaneously break the DNA at manypathways, unrelated where genes a short30. However, 6-7 base a “seedlarge region”element definesof this OTEspecificity pathway of suppression, derives from giving translational rise to a risk suppression, of affecting genomic loci that encodes the lncRNA. Proof-of-concept for two specific locations, excising out a large fragment of the this technique successfully knocked out lncRNA-21A, UCA1 CRISPR methods likewise have a risk of OTEs. Use of direct and AK023948 by removing large fragments ranging from ribonucleoproteinwhich, by definition, (RNP) cannot gene directly editing affect methods lncRNA function. (where 24 475 bp (lnRNA-21A) to 5.6 kb (UCA1) . A second strategy Cas9 protein and guide RNAs alone are employed in a DNA- uses CRISPR/Cas9 to engineer an RNA destabilizing element (such as a poly(A) signal, a miRNA binding site, a self- that employ -based overexpression of the Cas9 and cleaving , etc.) into the genomic sequence, causing guidefree format) RNAs, somewhat show far mitigating lower OTE the profiles OTEs risk than31 . methods Further, increased lncRNA transcript turnover/degradation. Using reduced risk of acting at off-target sites32. Hence, use of et al developed a MALAT1 knockout model by integrating improvedCas9 mutants ‘newer have generation been developed CRISPR that methods’ have a significantly can lower this strategy initially with zinc fingers , Gutschner a poly(A) signal upstream of the transcriptional start site, the risk of an OTE affecting results. As an additional 25,26 yielding a >1000 fold reduction of MALAT1 levels . The concern, CRISPR methods that permanently alter the DNA of a cell often result in the creation of different mutations on this approach. CRISPR-inhibition (CRISPRi) is a technique each chromosome in a cell or between cells in a population. thatadvent either of the uses CRISPR/Cas9 a catalytically technology inactive greatly (dead) simplified Cas9 It may be necessary to isolate clonally pure cell lines for (dCas9) protein to block RNA polymerase function, or, if higher transcriptional repression is desired, couples the kinds of genome editing experiments. dCas9 with a transcriptional repressor (such as KRAB). study before definitive conclusions can be made from these Gilbert et al targeted six different lncRNAs using dCas9- The discovery of thousands of lncRNAs and their KRAB, achieving >80% knockdown for 5/6 lncRNAs27. ambiguous roles in cellular processes has increased the In particular, effective use of CRISPRi methods requires need to have reliable knockdown techniques to help that the location of enhancer/promotor elements are determine their individual functions. Being able to select known and also if these regulatory elements solely control the knockdown method most likely to give the best results expression of the lncRNA or also contribute to expression saves time and money, and further progresses lncRNA of other (coding) transcripts. Using any CRISPR method, research. Here we described a repertoire of techniques transcripts that overlap, are transcribed from the opposite that can be used depending on lncRNA localization, strand, or are further processed into different isoforms lncRNA transcript (in)accessibility to , and the could also inadvertently be knocked out, confounding data transcriptional landscape within which they reside.

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