Aptazyme-Embedded Guide Rnas For Genome Editing

In few years ago, researches in synthetic biology and biotechnology have evolved rapidly and their application for bioengineering are tangible breakthroughs achieved nowadays to meet human needs. CRISPR technology is among current useful biotechnology tool used in prokaryotic and eukaryotic organisms for gene editing intention even though they are some challenges remained to alleviate.

Very recently, a combination of riboswitch and CRISPR development technology was launched with the aim to functionalize synthetic sgRNA designs to enable inducible and spatiotemporal regulation of CRISPR-based genetic editors in response to cellular or extracellular stimuli. Interestingly, some riboswitches can be developed and adapted to control sgRNA activity by adding corresponding sequences to its 5'end to repress the guide sequence in the unbound confirmation and reconstitute the active confirmation after ligand binding. In this sense, current study on genome editing and transcriptional activation in mammalian cells was able to incorporate ligand-responsive self-cleaving catalytic RNAs (aptazymes) into guide RNAs resulting a developed set of aptazyme-embedded guide RNAs that enable small molecule-controlled nuclease-mediated genome editing and small molecule-controlled base editing, as well as small molecule-dependent transcriptional activation in mammalian cells (Tang et al. 2017).

In addition, the advantage of using sgRNA-based inducible systems for synthetic biology applications has been recently showcased in a study demonstrating the ability to rewire cellular pathways by CRISPR-TR with modified sgRNAs containing ligand-responsive riboswitches. These sgRNA ‘signal conductors’ employ a strand-displacement mechanism to transition between OFF and ON states and can be coupled to a variety of inducers and dCas9 effectors (Liu et al. 2016). Furthermore, aptamer-controlled hammerhead ribozymes (so-called aptazymes) have been shown to be a versatile platform for the engineering of novel gene regulators. However, the generation of these novel aptazymes requires a functional aptamer–ribozyme connection, which can be difficult to engineer. Interestingly, Markus et. al, defined and elaborated novel methods and protocols for FACS-mediated aptazyme identification in bacteria and for engineering an aptazyme-based gene control system in mammalian cells (Wieland et al. 2012). Furthermore, Roßmanith and Narberhaus (2016) established three riboswitch-RNAT systems conferring dual regulation of transcription and translation depending on the two triggers ligand binding and temperature. This concept was based on designing ‘thermoswitches’ by integration of a thermosensor into riboswitches from different classes (TPP and lysine) as well as synthetic riboswitches (theophylline) in modular ways to gain functional regulatory elements. These ‘thermoswitches’ respond to the cognate ligand at low temperatures and are turned into a continuous on-state by a temperature upshift. These findings might help scientists to deplore riboswitches and RNATs for engineering synthetic RNA regulators due to their modular behavior Most recently, engineered drug-inducible catalytically inactive Cpf1 nuclease has been used to effectively construct transcriptional repressors in bacteria and plants.

Besides, when it is fused to transcriptional activation domains, the construct enabled to tune the expression of endogenous genes in human cells. It is in this sense that, a programmable ligand-controlled dAsCpf1 systems either by coupling crRNAs with engineered riboswitches or by fusing dAsCpf1 proteins with G protein-coupled receptors was constructed. These approaches using tunable CRISPR–Cpf1-based transcription factors allow the regulation of the transcription of endogenous genes in response to diverse classes of ligands, thus constructing artificial signaling pathways with rewired cellular input–output behaviors (Liu et al. 2017; Tak et al. 2017). Referring to a crucial impact of environmental pH on metabolism and behavior of living cells, Pham et al. (2017) engineered a set of riboswitch-based pH-sensing genetic devices to allow the control of gene expression. A digital pH sensing system designed can use the analogue-sensing characteristic of these devices for high resolution recording of host cell exposure to the external pH levels. This valuable innovation could be used in multiple engineering aspects of host cell for improved tolerance to a narrow range of organic acids, bioremediation and a precious phenotype for metabolic engineering.