Efficient base editing in tomato using a highly expressed transient system.

KEY MESSAGE: Base editing in tomatoes was achieved by transient expression. The Solanaceae plants, particularly the tomato (Solanum lycopersicum), is of huge economic value worldwide. The tomato is a unique model plant for studying the functions of genes related to fruit ripening. Deeper understanding of tomatoes is of great importance for both plant research and the economy. Genome editing technology, such as CRISPR/Cas9, has been used for functional genetic research. However, some challenges, such as low transformation efficiency, remain with this technology. Moreover, the foreign Cas9 and gRNA expression cassettes must be removed to obtain null-segregants In this study, we used a high-level transient expression system to improve the base editing technology. A high-level transient expression system has been established previously using geminiviral replication and a double terminator. The pBYR2HS vector was used for this transient expression system. nCas9-CDA and sgRNA-SlHWS were introduced into this vector, and the protein and RNA were then transiently expressed in tomato tissues by agroinfiltration. The homozygous mutant produced by base editing was obtained in the next generation with an efficiency of about 18%. nCas9-free next-generation plants were 71%. All the homozygous base-edited plants in next generation are nCas9-free. These findings show that the high-level transient expression system is useful for base editing in tomatoes.

Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells.

Synthetic biology has great potential for future therapeutic applications including autonomous cell programming through the detection of protein signals and the production of desired outputs. Synthetic RNA devices are promising for this purpose. However, the number of available devices is limited due to the difficulty in the detection of endogenous proteins within a cell. Here, we show a strategy to construct synthetic mRNA devices that detect endogenous proteins in living cells, control translation and distinguish cell types. We engineered protein-binding aptamers that have increased stability in the secondary structures of their active conformation. The designed devices can efficiently respond to target proteins including human LIN28A and U1A proteins, while the original aptamers failed to do so. Moreover, mRNA delivery of an LIN28A-responsive device into human induced pluripotent stem cells (hiPSCs) revealed that we can distinguish living hiPSCs and differentiated cells by quantifying endogenous LIN28A protein expression level. Thus, our endogenous protein-driven RNA devices determine live-cell states and program mammalian cells based on intracellular protein information.

Programmable mammalian translational modulators by CRISPR-associated proteins.

Translational modulation based on RNA-binding proteins can be used to construct artificial gene circuits, but RNA-binding proteins capable of regulating translation efficiently and orthogonally remain scarce. Here we report CARTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Gene control) to repurpose Cas proteins as translational modulators in mammalian cells. We demonstrate that a set of Cas proteins efficiently and orthogonally repress or activate the translation of designed mRNAs that contain a Cas-binding RNA motif in the 5'-UTR. By linking multiple Cas-mediated translational modulators, we designed and built artificial circuits like logic gates, cascades, and half-subtractor circuits. Moreover, we show that various CRISPR-related technologies like anti-CRISPR and split-Cas9 platforms could be similarly repurposed to control translation. Coupling Cas-mediated translational and transcriptional regulation enhanced the complexity of synthetic circuits built by only introducing a few additional elements. Collectively, CARTRIDGE has enormous potential as a versatile molecular toolkit for mammalian synthetic biology.

Synthetic RNA-based post-transcriptional expression control methods and genetic circuits.

RNA-based synthetic genetic circuits provide an alternative for traditional transcription-based circuits in applications where genomic integration is to be avoided. Incorporating various post-transcriptional control methods into such circuits allows for controlling the behaviour of the circuit through the detection of certain biomolecular inputs or reconstituting defined circuit behaviours, thus manipulating cellular functions. In this review, recent developments of various types of post-transcriptional control methods in mammalian cells are discussed as well as auxiliary components that allow for the creation and development of mRNA-based switches. How such post-transcriptional switches are combined into synthetic circuits as well as their applications in biomedical and preclinical settings are also described. Finally, we examine the challenges that need to be surmounted before RNA-based synthetic circuits can be reliably deployed into clinical settings.

Light-controllable RNA-protein devices for translational regulation of synthetic mRNAs in mammalian cells.

The photo-regulation of transgene expression is one effective approach in mammalian synthetic biology due to its high spatial and temporal resolution. While DNAs are mainly used as vectors, modified RNAs (modRNAs) are also useful for medical applications of synthetic biology, because they can avoid insertional mutagenesis and immunogenicity. However, the optogenetic control of modRNA-delivered transgenes is much more difficult than that of DNA-delivered transgenes. Here, we develop two types of photo-controllable translational activation systems that are compatible with modRNAs. One is composed of a heterodimerization domain-fused split translational activator protein and a photocaged heterodimerizer. The other is composed of a destabilizing domain-fused translational activator protein and a photocaged stabilizer. The destabilized type can be used for not only translational activation but also translational repression of the modRNAs. These photo-controllable translation systems will expand the application of mammalian synthetic biology research.

Orthogonal Protein-Responsive mRNA Switches for Mammalian Synthetic Biology.

The lack of available genetic modules is a fundamental issue in mammalian synthetic biology. Especially, the variety of genetic parts for translational control are limited. Here we report a new set of synthetic mRNA-based translational switches by engineering RNA-binding proteins (RBPs) and RBP-binding RNA motifs (aptamers) that perform strong translational repression. We redesigned the RNA motifs with RNA scaffolds and improved the efficiency of the repression to target RBPs. Using new and previously reported mRNA switches, we demonstrated that the orthogonality of translational regulation was ensured among five different RBP-responsive switches. Moreover, the new switches functioned not only with plasmid introduction, but also with RNA-only delivery, which provides a transient and safer regulation of expression. The translational regulators using RNA-protein interactions provide an alternative strategy to construct complex genetic circuits for future cell engineering and therapeutics.

RNA and protein-based nanodevices for mammalian post-transcriptional circuits.

Mammalian synthetic gene circuits have promise in biological and medical research due to their capability of controlling cellular functions. Especially, post-transcriptional circuits are growing in interest because of features that include compatibility and superior safety. RNA-based molecular nanodevices are often a core component in these circuits. RNA nanodevices that act as translational controllers should be suitable for designing genetic circuits that execute complex functions. In this review, we introduce recent progress in designing synthetic RNA-based circuitry and building mammalian post-transcriptional networks.

Author Correction: Synthetic RNA-based logic computation in mammalian cells.

The original version of this Article contained an error in the fourth sentence of the second paragraph of the 'Improving the performance of miRNA-responsive circuits' section of the Results, which incorrectly read 'We confirmed a significant fold-change between ON and OFF states (from 3.5- to 9.0-fold) in 293FT cells (Supplementary Figure 3).' The correct version states '4.6' in place of '3.5'. This has been corrected in both the PDF and HTML versions of the Article.The original version of the Supplementary Information contained a corresponding error in Supplementary Figure 3. The HTML has been updated to include a corrected version of the Supplementary Information.

Synthetic RNA-based logic computation in mammalian cells.

Synthetic biological circuits are designed to regulate gene expressions to control cell function. To date, these circuits often use DNA-delivery methods, which may lead to random genomic integration. To lower this risk, an all RNA system, in which the circuit and delivery method are constituted of RNA components, is preferred. However, the construction of complexed circuits using RNA-delivered devices in living cells has remained a challenge. Here we show synthetic mRNA-delivered circuits with RNA-binding proteins for logic computation in mammalian cells. We create a set of logic circuits (AND, OR, NAND, NOR, and XOR gates) using microRNA (miRNA)- and protein-responsive mRNAs as decision-making controllers that are used to express transgenes in response to intracellular inputs. Importantly, we demonstrate that an apoptosis-regulatory AND gate that senses two miRNAs can selectively eliminate target cells. Thus, our synthetic RNA circuits with logic operation could provide a powerful tool for future therapeutic applications.

Protein-driven RNA nanostructured devices that function in vitro and control mammalian cell fate.

Nucleic acid nanotechnology has great potential for future therapeutic applications. However, the construction of nanostructured devices that control cell fate by detecting and amplifying protein signals has remained a challenge. Here we design and build protein-driven RNA-nanostructured devices that actuate in vitro by RNA-binding-protein-inducible conformational change and regulate mammalian cell fate by RNA-protein interaction-mediated protein assembly. The conformation and function of the RNA nanostructures are dynamically controlled by RNA-binding protein signals. The protein-responsive RNA nanodevices are constructed inside cells using RNA-only delivery, which may provide a safe tool for building functional RNA-protein nanostructures. Moreover, the designed RNA scaffolds that control the assembly and oligomerization of apoptosis-regulatory proteins on a nanometre scale selectively kill target cells via specific RNA-protein interactions. These findings suggest that synthetic RNA nanodevices could function as molecular robots that detect signals and localize target proteins, induce RNA conformational changes, and programme mammalian cellular behaviour.Nucleic acid nanotechnology has great potential for future therapeutic applications. Here the authors build protein-driven RNA nanostructures that can function within mammalian cells and regulate the cell fate.