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Dynamic RNA Nanotechnology Enters the CRISPR Toolbox KaHo Leung and Yamuna Krishnan Department of Chemistry and Grossman Institute of Neuroscience, Quantitative Biology and Human Behavior, The University of Chicago, Chicago, Illinois 60637, United States
showing that CRISPR could edit genomes in vivo and in eukaryotic cells.5,6 Studies on gRNAs revealed that Cas9 activity is tolerant of modifications in the crRNA:tracrRNA complementary region.7 This allows the engineering of gRNAs to facilitate the introduction of auxiliary components that enable the conditional control of gRNA activity via structural changes triggered by small molecules, nucleases, nuclease-recruiting DNA, or photoactivation.8 Here, Pierce and colleagues develop an endogenous RNA-triggered conditional guide RNA (cgRNA) that either turns on or turns off gene expression mediated by Cas9 function. In this system, Pierce uses catalytically dead Cas9 (dCas9) that blocks transcription of the DNA at the binding site.9 Pierce’s team first construct a cgRNA that has a site (blue) containing an extended loop that acts as a binding site for an RNA trigger (Figure 1c). In the absence of the RNA trigger, the cgRNA is capable of complexing dCas9 and guiding it to the target gene where it represses transcription. On the contrary, a complementary RNA trigger (blue) will hybridize with this loop region in the cgRNA resulting in an overall structure that cannot complex with dCas9. This results in the target gene being efficiently transcribed. The performance of the cgRNA is evaluated in Escherichia coli that express dCas9, which is targeted to a reporter gene encoding a fluorescent protein. The presence of the cognate RNA trigger enhances protein expression by ∼3−6 fold. The authors
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Controlling CRISPR/Cas function with endogenous RNA triggers.
T
he discovery of homologous recombination led to the knowledge that introducing a double-strand DNA break at a designated site in the genome enhances targeted gene integration.1 This spurred a plethora of strategies, such as meganucleases, zinc fingers, and TALENS, to selectively introduce double-strand DNA breaks in designated locations at genomes.2 The most recent technology, clustered regularly interspaced short palindromic repeats - CRISPR-associated protein 9 (CRISPR-Cas9), edits genomes based on a guide RNA (gRNA) that targets Cas9, a DNA endonuclease, to specific sites in genomic DNA, thus enabling the addition, elimination, or alteration of DNA sequences at the targeted site in the genome.2 It has revolutionized our ability to edit the genome of virtually any organism. In this issue of ACS Central Science, Pierce and colleagues describe an elegant method to program CRISPR/Cas function by engineering conditional guide RNAs (cgRNAs) whose activity is triggered by the presence of an endogenous RNA trigger.3
It has revolutionized our ability to edit the genome of virtually any organism.
Although biomedical applications seem a little far-fetched at present, the capacity to achieve tissue-specific silencing conditionally would prove particularly powerful to study the role of noncoding RNAs or RNA modifications in organism development.
Gene editing based on CRISPR-Cas technology requires two short RNAs, a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA), that together form a trimolecular complex with Cas9 (Figure 1a). The complexed crRNA now guides the endonuclease Cas9 to a specific site in the genome that is complementary to the 5′ end of the crRNA where Cas9 introduces a double strand break (Figure 1a). Doudna, Charpentier, and co-workers combined the crRNA and tracrRNA to give a single chimeric RNA popularly known now as gRNA (Figure 1b).4 Several groundbreaking studies subsequently emerged © 2019 American Chemical Society
Published: June 18, 2019 1111
DOI: 10.1021/acscentsci.9b00550 ACS Cent. Sci. 2019, 5, 1111−1113
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Figure 1. Schematic illustration of the CRISPR/Cas system. Cas9 can be guided by (a) a two-RNA structure formed by trans-activating crRNA (tracrRNA) and CRISPR RNA (crRNA) or by (b) single guide RNA (gRNA) to cleave the specific site in genome. Schematic illustration of the mechanism of Pierce and colleagues’ (c) terminator switch cgRNA and (d) toehold switch cgRNA controlling CRISPR/Cas function with endogenous RNA triggers.3
Although there is a previous description of cgRNAs responsive to an RNA trigger by Siu et al, the RNA trigger turns off target gene expression.10 In the present study, the presence of an RNA trigger enhances gene expression and can also be engineered to shut off gene expression. Further, the strategy described herein is broadly applicable and demonstrated in both bacteria as well as mammalian cells. While the ability to control dCas9 function in response to endogenous RNA triggers is an extraordinary addition to the CRISPR toolbox, a few outstanding challenges remain. The fold change in gene expression in mammalian cells is much lower than that achieved in bacteria, only ∼2−5 fold change. This is likely due to potentially higher crosstalk of the
then modified the loop length and the number of loops on the cgRNA to further optimize the change in gene expression levels and improve it to ∼18-fold higher expression. This was made possible by a cgRNA design that can enhance dCas9 activity such that gene expression was more effectively shut off. In a final example, Pierce and colleagues demonstrate the reverse logic, where, in the absence of the RNA trigger, the cgRNA is incapable of guiding dCas9 to its target site, which leads to high expression of a target gene. However, in the presence of an RNA trigger, the cgRNA the targeting sequence on the cgRNA is now unmasked, and dCas9 localizes to the target gene, preventing its transcription (Figure 1d). 1112
DOI: 10.1021/acscentsci.9b00550 ACS Cent. Sci. 2019, 5, 1111−1113
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cgRNA with other cellular RNA molecules. New cgRNA designs that show lower crosstalk with other RNA sequences would be required for greater effectiveness in eukaryotic systems as well as to toggle the expression of multiple genes in concert. Although biomedical applications seem a little far-fetched at present, the capacity to achieve tissue-specific silencing conditionally would prove particularly powerful to study the role of noncoding RNAs or RNA modifications in organism development. Author Information
E-mail:
[email protected]. ORCID
Yamuna Krishnan : 0000-0001-5282-8852 Yamuna Krishnan: 0000-0001-5282-8852 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Y.K. acknowledges the Mergel Funsky Award and the Women’s Board of the University of Chicago for support. K.L. is supported by the Orphan Disease Center, part of the University of Pennsylvania’s School of Medicine.
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REFERENCES REFERENCES (1) Orr-Weaver, T. L.; Szostak, J. W.; Rothstein, R. J. Yeast Transformation: a Model System for the Study of Recombination. Proc. Natl. Acad. Sci. U. S. A. 1981, 78, 6354−6358. (2) Knott, G. J.; Doudna, J. A. CRISPR-Cas Guides the Future of Genetic Engineering. Science 2018, 361, 866−869. (3) Hanewich-Hollatz, M. H.; Chen, Z.; Hochrein, L. M.; Huang, J.; Pierce, N. A. Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacterial and Mammalian Cells via Dynamic RNA Nanotechnology. ACS Cent. Sci. 2019, DOI: 10.1021/acscentsci.9b00340. (4) Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A.; Charpentier, E. A Programmable dual-RNA-guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 2012, 337, 816−821. (5) Jinek, M.; East, A.; Cheng, A.; Lin, S.; Ma, E.; Doudna, J. RNAprogrammed Genome Editing in Human Cells. eLife 2013, 2, No. e00471. (6) Cong, L.; Ran, F. A.; Cox, D.; Lin, S.; Barretto, R.; Habib, N.; Hsu, P. D.; Wu, X.; Jiang, W.; Marraffini, L. A.; et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 2013, 339, 819−823. (7) Briner, A. E.; Donohoue, P. D.; Gomaa, A. A.; Selle, K.; Slorach, E. M.; Nye, C. H.; Haurwitz, R. E.; Beisel, C. L.; May, A. P.; Barrangou, R. Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality. Mol. Cell 2014, 56, 333−339. (8) Adli, M. The CRISPR Tool Kit for Genome Editing and Beyond. Nat. Commun. 2018, 9, 1911. (9) Qi, L. S.; Larson, M. H.; Gilbert, L. A.; Doudna, J. A.; Weissman, J. S.; Arkin, A. P.; Lim, W. A. Repurposing CRISPR as an RNA-guided Platform for Sequence-specific Control of Gene Expression. Cell 2013, 152, 1173−1183. (10) Siu, K. H.; Chen, W. Riboregulated Toehold-gated gRNA for Programmable CRISPR-Cas9 Function. Nat. Chem. Biol. 2019, 15, 217−220.
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DOI: 10.1021/acscentsci.9b00550 ACS Cent. Sci. 2019, 5, 1111−1113
