RNA Switches Using Cas Proteins.

Expanding the number of available RNA-binding proteins (RBPs) is vital to establishing posttranscriptional circuits in mammalian cells. We focused on CRISPR-Cas systems and exploited Cas proteins for their versatility as RBPs. The translation of genes encoded in an mRNA becomes regulatable by a Cas protein by inserting a crRNA/sgRNA sequence recognizable by the specific Cas protein into its 5'UTR. These Cas protein-responsive switches vastly expand the available tools in synthetic biology because of the wide range of Cas protein orthologs that can be used as trigger proteins.Here, we describe the design principle of Cas protein-responsive switches, both plasmid and RNA versions, using Streptococcus pyogenes Cas9 (SpCas9) as an example and show an example of its use in mammalian cells, HEK293FT cells.

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.

A versatile and robust cell purification system with an RNA-only circuit composed of microRNA-responsive ON and OFF switches.

Human induced pluripotent stem cells (iPSCs) are promising cell resources for cell therapy and drug discovery. However, iPSC-derived differentiated cells are often heterogenous and need purification using a flow cytometer, which has high cost and time consumption for large-scale purification. MicroRNAs (miRNAs) can be used as cell selection markers, because their activity differs between cell types. Here, we show miRNA-responsive ON and OFF switch mRNAs for robust cell purification. The ON switch contains a miRNA-target sequence after the polyadenylate tail, triggering translational activation by sensing the target miRNA. By designing RNA-only circuits with miRNA-ON and -OFF switch mRNAs that encode a lethal ribonuclease, Barnase, and its inhibitor, Barstar, we efficiently purified specific cell types, including human iPSCs and differentiated cardiomyocytes, without flow cytometry. Synthetic mRNA circuits composed of ON and OFF switches provide a safe, versatile, and time-saving method to purify various cell types for biological and clinical applications.

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.

Cell-Type-Specific CRISPR Activation with MicroRNA-Responsive AcrllA4 Switch.

Anti-CRISPR proteins have the potential to regulate CRISPR-Cas systems in a cell-type-specific manner. To selectively edit the genome in target cells, we controlled the expression of AcrllA4, a Streptococcus pyogenes Cas9 inhibitor, based on endogenous microRNA (miRNA) activity. We designed a miRNA-responsive AcrllA4 switch, which is a synthetic mRNA that contains a completely complementary sequence to an arbitrary miRNA at the 5'-UTR region and encodes AcrllA4. Together with the Cas9- or dCas9-VPR-guide RNA complex, this switch functions as a cell-specific Cas9 or dCas9-VPR activator that induces gene knockout or activation depending on the target miRNA. By sensing intracellular miRNAs, the conditional CRISPR-Cas9 ON system that we report could provide a powerful tool for future therapeutic applications and genome engineering.

Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch.

The CRISPR-Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR-Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information.