As the best-known synthetic biology system, the CRISPR-Cas9 system is a practical tool for site-directed mutation and the identification of gene function. Until now, several CRISPR circuits have been created in bacterial, yeast and mammalian cells (figure), and many of them constitute experimental demonstrations of the efficiency of the stratification and integration of signals provided by the CRISPR tools. For the same reason, the orthogonality of CRISPR is practically unlimited, with a theoretical upper limit of 420 possible 20-nt sequences. In CRISPR circuits, the binding of the effector protein (almost always dCas9 or a derivative) to DNA is mediated by an associated RNA molecule, gRNA, and this effectively avoids many of the intrinsic limitations of TFs.
One of the applications in which the use of CRISPR tools has enormous potential is the construction of synthetic gene circuits. With the emergence of new classes of programmable genetic tools, in particular, the establishment and optimization of CRISPR and associated technological platforms, synthetic biology and its vital field, synthetic genomics, are entering a new era of greater possibilities. In short, PAM refers to a short sequence that resides in the elements of the exogenous nucleic acid (usually at the 3' end of the target DNA), but not in the CRISPR matrix and its guide RNAs, which make it possible to discriminate the ingredients of nucleic acids that are their own and not typical of microbes. It is to be expected that the CRISPR toolkits will be of particular importance for the future of synthetic genomics because of their great potential to open up new ways of manipulating and expressing genetic information, which will undoubtedly greatly transform synthetic genomics and biology.
The greater knowledge of the CRISPR classifications and their mechanisms of action opens up new areas of application in the synthetic genome of an organism through these systems. Despite some limitations that need to be overcome, the arrival of CRISPR techniques has undoubtedly created a new era for genomic engineering in yeasts. While the dynamic range of TFs is generally high, that of the CRISPRI and CRISPRA systems seems to depend largely on the particular implementation. The CRISPRlator showed a strong inheritance of the oscillatory state in all cell divisions, causing long-term synchronous oscillations of a cell population in a microfluidic chamber.
When an exogenous nucleic acid invades the cell, its identity is recorded in a particular group of the host genome (the CRISPR matrix) in the form of short stretches of the invading sequence. CRISPR-mediated gene expression control is extremely programmable and easy to design; modifying the 20 nt spacer sequence of gRNA is sufficient to direct dCas9 activity to a new target. As CRISPR systems continue to be discovered, a variety of programmable nucleases have joined the ranks of genome editing. Therefore, the choice of CRISPR technology as a framework for the design of synthetic circuits constitutes a valid alternative to complement or replace TFs in synthetic circuits and promises the realization of more ambitious designs.
