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• W2999500098 abstract "Live-cell RNA imaging is a powerful approach to observe the real-time dynamics of RNA metabolism. Two recent papers describe an optimized fluorescence-based CRISPR-Cas13 approach to image colocalized or repeat-containing RNAs in real time, as well as demonstrate simultaneous RNA-DNA labeling by using Cas13 and Cas9 in tandem. Live-cell RNA imaging is a powerful approach to observe the real-time dynamics of RNA metabolism. Two recent papers describe an optimized fluorescence-based CRISPR-Cas13 approach to image colocalized or repeat-containing RNAs in real time, as well as demonstrate simultaneous RNA-DNA labeling by using Cas13 and Cas9 in tandem. At the subcellular level, RNA transcripts are dynamically localized, and this spatiotemporal organization can regulate gene function (Chen, 2016Chen L.L. Linking Long Noncoding RNA Localization and Function.Trends Biochem. Sci. 2016; 41: 761-772Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar). Several different fluorescent tools exist to visualize RNA localization dynamics, including the MS2-MCP system, molecular beacons, and fluorogenic RNA aptamers; however, each have limitations that make it difficult to readily track individual RNA species in a live cell. For example, although the MS2-MCP approach has worked quite well for single mRNA tracking, the approach requires the exogenous insertion of ∼24 MS2 hairpins into the RNA of interest, which can be not only cumbersome but can also affect the structure and function of the tagged RNA (Tutucci et al., 2018Tutucci E. Livingston N.M. Singer R.H. Wu B. Imaging mRNA In Vivo, from Birth to Death.Annu. Rev. Biophys. 2018; 47: 85-106Crossref PubMed Scopus (74) Google Scholar). More recently, CRISPR-Cas-based approaches, including RNA-targeting CRISPR-Cas9, have been explored to track the bulk movement of highly abundant mRNAs, such as β-actin to stress granules, but it is not yet clear whether visualizing other, less abundant RNAs or single transcripts is possible (Nelles et al., 2016Nelles D.A. Fang M.Y. O'Connell M.R. Xu J.L. Markmiller S.J. Doudna J.A. Yeo G.W. Programmable RNA tracking in live cells with CRISPR/Cas9.Cell. 2016; 165: 488-496Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). Therefore, there is still a need for high signal-to-noise ratio (S/N), easily programmable, and less invasive methods to visualize endogenous (untagged) single-RNA transcripts in live cells. Two recent papers, Yang et al., 2019Yang L.-Z. Wang Y. Li S.-Q. Yao R.-W. Luan P.-F. Wu H. Carmichael G.G. Chen L.-L. Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems.Mol. Cell. 2019; 76: 981-997.e7Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar and Wang et al., 2019Wang H. Nakamura M. Abbott T.R. Zhao D. Luo K. Yu C. Nguyen C.M. Lo A. Daley T.P. La Russa M. et al.CRISPR-mediated live imaging of genome editing and transcription.Science. 2019; 365: 1301-1305Crossref PubMed Scopus (127) Google Scholar, overcome some of these hurdles and describe an optimized fluorescence-based CRISPR-Cas13 approach to image colocalized or repeat-containing RNAs. CRISPR-Cas13 is a family of bacterial RNA-guided single-stranded-RNA (ssRNA)-targeting ribonucleases that can be divided into four subtypes: Cas13a–Cas13d. With similar target specificity to CRISPR-Cas9, Cas13 can be programmed to degrade unique RNA sequences within the transcriptome (Abudayyeh et al., 2017Abudayyeh O.O. Gootenberg J.S. Essletzbichler P. Han S. Joung J. Belanto J.J. Verdine V. Cox D.B.T. Kellner M.J. Regev A. et al.RNA targeting with CRISPR-Cas13.Nature. 2017; 550: 280-284Crossref PubMed Scopus (961) Google Scholar), and catalytically deactivated Cas13 (dCas13) orthologs fused to a range of effector domains can be used to perform RNA sequence-specific functions, such as A-to-I editing, modulating translation, or splicing (Cox et al., 2017Cox D.B.T. Gootenberg J.S. Abudayyeh O.O. Franklin B. Kellner M.J. Joung J. Zhang F. RNA editing with CRISPR-Cas13.Science. 2017; 358: 1019-1027Crossref PubMed Scopus (863) Google Scholar, Rauch et al., 2018Rauch S. He C. Dickinson B.C. Targeted m6A Reader Proteins To Study Epitranscriptomic Regulation of Single RNAs.J. Am. Chem. Soc. 2018; 140: 11974-11981Crossref PubMed Scopus (64) Google Scholar, Konermann et al., 2018Konermann S. Lotfy P. Brideau N.J. Oki J. Shokhirev M.N. Hsu P.D. Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors.Cell. 2018; 173: 665-676.e14Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). To date, however, only GFP-tagged dCas13a from Leptotrichia wadei (dLwaCas13a) has been used for imaging stress-induced bulk β-actin mRNA movement (Abudayyeh et al., 2017Abudayyeh O.O. Gootenberg J.S. Essletzbichler P. Han S. Joung J. Belanto J.J. Verdine V. Cox D.B.T. Kellner M.J. Regev A. et al.RNA targeting with CRISPR-Cas13.Nature. 2017; 550: 280-284Crossref PubMed Scopus (961) Google Scholar). As such, it has yet to be seen whether any of the increasingly large number of discovered Cas13 orthologs are more amenable for RNA imaging applications. To address this, Yang et al., 2019Yang L.-Z. Wang Y. Li S.-Q. Yao R.-W. Luan P.-F. Wu H. Carmichael G.G. Chen L.-L. Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems.Mol. Cell. 2019; 76: 981-997.e7Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar cleverly utilized NEAT1-long-noncoding RNA (lncRNA) paraspeckle colocalization to screen eight fluorescent-protein-tagged dCas13 orthologs for their RNA imaging prowess. NEAT1 is a moderately expressed lncRNA that is a spatially restricted, integral structural component of paraspeckles: ∼300 nm subnuclear bodies that comprise ∼50 copies of NEAT1 and a swath of RNA-binding proteins (e.g., NONO). As such, it is a useful tool for RNA-imaging screening purposes, because the colocalization of multiple NEAT1 molecules allows for sufficient S/N discrimination above a fluorescent background, and observing NONO colocalization provides some measure of specificity (Figure 1A). Of all eight Cas13 orthologs tested, only dPspCas13b and dPguCas13b yielded sufficient S/N and specificity to visualize NEAT1-containing paraspeckles. Interestingly, while dRfxCas13d had a comparable on-target signal, significant non-specific accumulation with NONO was observed when using a non-targeting guide-RNA (gRNA) control. For all other Cas13 orthologs that Yang et al., 2019Yang L.-Z. Wang Y. Li S.-Q. Yao R.-W. Luan P.-F. Wu H. Carmichael G.G. Chen L.-L. Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems.Mol. Cell. 2019; 76: 981-997.e7Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar tested, they primarily exhibited either no NEAT1 localization or aberrant nucleolar localization. Why may this be? We speculate that this may be due to either insufficient Cas13 gRNA loading or gRNA expression in combination with inherent non-specific RNA binding of apo-Cas13 to rRNA generated in the nucleolus. This raises several questions. Why do some Cas13 orthologs behave better than others? Does the observed behavior of these Cas13 orthologs have implications for other Cas13 applications? These observations should certainly pique the interest of budding Cas13 aficionados. In contrast to the difficulties encountered with dRfxCas13d by Yang et al., 2019Yang L.-Z. Wang Y. Li S.-Q. Yao R.-W. Luan P.-F. Wu H. Carmichael G.G. Chen L.-L. Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems.Mol. Cell. 2019; 76: 981-997.e7Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, Wang et al., 2019Wang H. Nakamura M. Abbott T.R. Zhao D. Luo K. Yu C. Nguyen C.M. Lo A. Daley T.P. La Russa M. et al.CRISPR-mediated live imaging of genome editing and transcription.Science. 2019; 365: 1301-1305Crossref PubMed Scopus (127) Google Scholar successfully utilized RfxCas13d in tandem with dCas9 to simultaneously label RNA and DNA in live cells (Figure 1B). Here, by electroporating a Cas13 gRNA and a Cas9:gRNA complex into stably expressing dRfxCas13d cells, the authors were able to visualize both the transcription of a MS2-repeat-tagged mRNA and also the genomic locus from which the tagged mRNA emanates in real time. Interestingly, non-specific dRfxCas13d binding was not directly observed, most likely because of the fluorescent-labeling approach used. Here, rather than labeling dRfxCas13d with a fluorescent protein, Wang et al., 2019Wang H. Nakamura M. Abbott T.R. Zhao D. Luo K. Yu C. Nguyen C.M. Lo A. Daley T.P. La Russa M. et al.CRISPR-mediated live imaging of genome editing and transcription.Science. 2019; 365: 1301-1305Crossref PubMed Scopus (127) Google Scholar directly labeled the gRNA with a fluorescent dye (ATTO-647). Given the instability of a free fluorescently labeled gRNA in the live cell (as also shown by Wang et al., 2019Wang H. Nakamura M. Abbott T.R. Zhao D. Luo K. Yu C. Nguyen C.M. Lo A. Daley T.P. La Russa M. et al.CRISPR-mediated live imaging of genome editing and transcription.Science. 2019; 365: 1301-1305Crossref PubMed Scopus (127) Google Scholar), it can be inferred that the dRfxCas13d:gRNA and/or dRfxCas13d:gRNA:RNA-target complexes are the predominate species being observed. These observations suggest that direct gRNA labeling may circumvent some of the specificity issues associated with Cas13-protein labeling and perhaps help to resurrect additional Cas13 orthologs for imaging applications. It should be noted that Yang et al., 2019Yang L.-Z. Wang Y. Li S.-Q. Yao R.-W. Luan P.-F. Wu H. Carmichael G.G. Chen L.-L. Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems.Mol. Cell. 2019; 76: 981-997.e7Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar also briefly demonstrated that the delivery of a precomplexed dPspCas13b and fluorescent gRNA using electroporation can be successfully used to image colocalized SatIII repeat RNAs. Together, these studies highlight that additional work optimizing Cas13-based imaging technologies may yield further improvements in imaging performance. We would also like to briefly highlight that Wang et al., 2019Wang H. Nakamura M. Abbott T.R. Zhao D. Luo K. Yu C. Nguyen C.M. Lo A. Daley T.P. La Russa M. et al.CRISPR-mediated live imaging of genome editing and transcription.Science. 2019; 365: 1301-1305Crossref PubMed Scopus (127) Google Scholar were able to optimize a DNA imaging approach using purified precomplexed Cas9 protein and fluorescently labeled gRNA for DNA imaging (dubbed CRISPR LiveFISH), and, in addition to the experiment described above, this approach was also used to track real-time dynamics of genome editing and chromosome translocation. However, we only focus on RNA imaging in this spotlight because of space constraints. Yang et al., 2019Yang L.-Z. Wang Y. Li S.-Q. Yao R.-W. Luan P.-F. Wu H. Carmichael G.G. Chen L.-L. Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems.Mol. Cell. 2019; 76: 981-997.e7Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar also went on to show that dPspCas13b and dPguCas13b can successfully image other RNAs beyond NEAT1, including an endogenous repeat-containing mRNA MUC4, a stress-induced SatIII repeat RNA, and an overexpressed GCN4-repeat-containing mRNA. This approach was also extended to achieve simultaneous, dual-color, multiplexed repeat-RNA imaging using dPspCas13b and dPguCas13b and, in a similar vein to Wang et al., 2019Wang H. Nakamura M. Abbott T.R. Zhao D. Luo K. Yu C. Nguyen C.M. Lo A. Daley T.P. La Russa M. et al.CRISPR-mediated live imaging of genome editing and transcription.Science. 2019; 365: 1301-1305Crossref PubMed Scopus (127) Google Scholar, dual-color repeat-DNA and repeat-RNA imaging by using dCas9 and dCas13, respectively. Overall, what caught our eye here in this set of experiments was that, depending on which fluorescent protein fusion was used and the configuration of nuclear localization sequences in the construct, PspCas13b exhibited vastly different (and sometimes nonintuitive) abilities to preferentially target nuclear versus cytoplasmically localized RNAs and that in some cases, as few as eight GCN4 repeats yielded a sufficient signal. This highlights that a number of Cas13 constructs may need to be trialed depending on the RNA target of interest and that targeting non-repeat RNAs might soon be within reach with further improvements in construct design, as well as the use of pre-gRNA arrays that can generate multiple unique gRNAs per single RNA target. Finally, Yang et al., 2019Yang L.-Z. Wang Y. Li S.-Q. Yao R.-W. Luan P.-F. Wu H. Carmichael G.G. Chen L.-L. Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems.Mol. Cell. 2019; 76: 981-997.e7Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar went on to use Cas13 to interrogate paraspeckle morphology and dynamics in real time. Previous work has shown that paraspeckle proteins are organized in different layers along the radial bundles of NEAT1 transcripts, thereby forming a core-shell spheroid structure (West et al., 2016West J.A. Mito M. Kurosaka S. Takumi T. Tanegashima C. Chujo T. Yanaka K. Kingston R.E. Hirose T. Bond C. et al.Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization.J. Cell Biol. 2016; 214: 817-830Crossref PubMed Scopus (181) Google Scholar), with core components sequestered toward the inside of the spheroid while other components are localized to the outside of the paraspeckle (Hirose et al., 2014Hirose T. Virnicchi G. Tanigawa A. Naganuma T. Li R. Kimura H. Yokoi T. Nakagawa S. Bénard M. Fox A.H. Pierron G. NEAT1 long noncoding RNA regulates transcription via protein sequestration within subnuclear bodies.Mol. Biol. Cell. 2014; 25: 169-183Crossref PubMed Scopus (297) Google Scholar). Using a combination Cas13-NEAT1 and NONO tagging, Yang et al., 2019Yang L.-Z. Wang Y. Li S.-Q. Yao R.-W. Luan P.-F. Wu H. Carmichael G.G. Chen L.-L. Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems.Mol. Cell. 2019; 76: 981-997.e7Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar observed the expected core-shell spheroid paraspeckle structure, differential core versus shell motions, and fusion and fission of paraspeckles in real time, leading the authors to suggest a kiss-and-run and fusion model for paraspeckle dynamics, in turn providing a new tool to study paraspeckle function in diverse biological processes, such as transcription regulation and stress response, and in diseases such as cancer (Fox et al., 2018Fox A.H. Nakagawa S. Hirose T. Bond C.S. Paraspeckles: Where Long Noncoding RNA Meets Phase Separation.Trends Biochem. Sci. 2018; 43: 124-135Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Despite optimization of these dCas13 imaging approaches, there is still room for further gains in performance. For example, not all gRNAs worked well, and even small changes in gRNA position resulted in significant changes in imaging performance. In contrast to Cas9, no predictive Cas13 gRNA target-site selection algorithms currently exist, so it is still a difficult task to select optimal gRNAs. Furthermore, most RNAs inconveniently don’t contain a sufficient number of sequence repeats to obtain the S/N required for imaging applications. It will be interesting to see whether non-repeat RNA imaging is possible through the use of pre-gRNA arrays and Cas13’s pre-gRNA processing ability to tile a target RNA with a large enough number of unique gRNAs for single-molecule RNA imaging. Regardless, these studies elegantly show the potential of using Cas13 to study live-cell dynamics and subcellular localization of repeat-containing RNA transcripts, as well as the ability to simultaneously visualize gene expression at the DNA and RNA level. This is a very exciting development for a burgeoning field. M.R.O. is an inventor on patents related to CRISPR-Cas systems and their various uses. M.R.O. is a scientific advisory board member of Dahlia Biosciences and Locana. Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 SystemsYang et al.Molecular CellNovember 19, 2019In BriefYang et al. show that the dCas13 system is capable of labeling RNAs. Applying orthogonal dCas13s or combining with dCas9 allows simultaneous visualization of RNA-RNA and DNA-RNA in living cells. The dCas13 system is user friendly in real-time RNA imaging without requiring genetic manipulation. Full-Text PDF Open Archive" @default.
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• W2999500098 title "Put on Your Para-spectacles: The Development of Optimized CRISPR-Cas13-Based Approaches to Image RNA Dynamics in Real Time" @default.
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