In This Issue pubs.acs.org/synthbio
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SWITCHING PROTEIN LOCALIZATION BY SITE-DIRECTED RNA EDITING UNDER CONTROL OF LIGHT
ENGINEERED CRISPR SYSTEMS FOR NEXT GENERATION GENE THERAPIES
During expression genetic information is diversified by various mechanisms. Even when encoded in a single genetic locus, many proteins occur in several isoforms, which result from alternative promotor usage or alternative splicing. Another way of
Gene therapy is now a viable therapeutic strategy for a range of clinical conditions. An ideal in vivo gene therapy platform provides safe, reprogrammable, and precise strategies which modulate cell and tissue gene regulatory networks with a high temporal and spatial resolution. Clustered regularly interspaced short palindromic repeats (CRISPR), a bacterial adoptive immune system, and its CRISPR-associated protein 9 (Cas9), have gained attention for the ability to target and modify DNA sequences on demand with unprecedented flexibility and precision. The precision and programmability of Cas9 is derived from its complexation with a guide-RNA (gRNA) that is complementary to a desired genomic sequence. CRISPR systems open-up widespread applications including genetic disease modeling, functional screens, and synthetic gene regulation. The plausibility of in vivo genetic engineering using CRISPR has garnered significant traction as a next generation in vivo therapeutic. However, there are hurdles that need to be addressed before CRISPR-based strategies are fully implemented. Some key issues center on the controllability of the CRISPR platform, including minimizing genomic-off target effects and maximizing in vivo gene editing efficiency, in vivo cellular delivery, and spatial-temporal regulation. The modifiable components of CRISPR systems: Cas9 protein, gRNA, delivery platform, and the form of CRISPR system delivered (DNA, RNA, or ribonucleoprotein) have recently been engineered independently to design a better genome engineering toolbox. In this issue, Pineda et al. (DOI: 10.1021/acssynbio.7b00011) evaluate CRISPR potential as a next generation in vivo gene therapy platform and discuss bioengineering advancements that can address challenges associated with clinical translation of this emerging technology.
diversification is a process called RNA editing. Site directed RNA editing is an engineered tool for the post-transcriptional manipulation of RNA and proteins. In this issue, Vogel et al. (DOI: 10.1021/acssynbio.7b00113) demonstrate the inclusion of additional N- and C-terminal protein domains in an RNA editing-dependent manner to switch between protein isoforms in mammalian cell culture. By inclusion of localization signals, a switch of the subcellular protein localization was achieved. This included the shift from the cytoplasm to the outer-membrane, which typically is inaccessible at the protein-level. Furthermore, the strategy allows implementation of the photocaging to achieve spatiotemporal control of isoform switching. The strategy does not require substantial genetic engineering, and might well complement current optogenetic and optochemical approaches. In combination with light-control, this tool could provide new opportunities to address biological questions in basic research. In the future, proteins might be steered to the cell surface by using light-activated RNA editing to manipulate intracellular signaling but also extracellular events such as cell−cell and cell−matrix interactions in a
Received: September 5, 2017 Published: September 15, 2017
spatiotemporal manner. © 2017 American Chemical Society
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DOI: 10.1021/acssynbio.7b00321 ACS Synth. Biol. 2017, 6, 1605−1606
ACS Synthetic Biology
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In This Issue
A FLUORESCENT SPLIT APTAMER FOR VISUALIZING RNA−RNA ASSEMBLY IN VIVO
RNA−RNA interactions drive key processes in biology, such as RNA interference, retroviral genome dimerization, CRISPR-Cas activation and targeting, and post-transcriptional regulation of RNA by long noncoding RNAs. RNA−RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA−RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In this issue, Alam et al. (DOI: 10.1021/ acssynbio.7b00059) present the binary “Split-Broccoli” system as a stand-alone RNA logic gate and as a device for monitoring RNA−RNA hybridization in vitro and in vivo. The design−build−test cycle iterates from in silico design to in vitro implementation and finally to in vivo functionality. The structure-based strategy begins with an unsplit dimeric aptamer within a stabilizing RNA architecture and continues with its bisection into a two-component system. The system assembles reliably in vitro when purified components are thermally renatured, and the addition of transcription terminator structures improves the “OFF” level of the system. At physiological temperature, in vitro assembly approximately follows second-order kinetics but appears to be limited by kinetic traps that prevent fast refolding into the functional hybrid. However, when individual RNAs of the system are cotranscribed in vitro, fluorescence signal strength roughly approximates the unsplit variant over a 4 h time course. The Split-Broccoli system also assembles and activates fluorescence when expressed in vivo, whether as a stand-alone AND gate or as a tool to monitor an RNA−RNA hybridization event that drives translation of a red fluorescent protein. The authors anticipate that the SplitBroccoli system will be an enabling addition to the toolkit for RNA biologists.
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DOI: 10.1021/acssynbio.7b00321 ACS Synth. Biol. 2017, 6, 1605−1606
