By Sashini Ranawana
When the first eukaryotic cells were subjected to CRISPR/Cas9- mediated genetic modifications in 2013, it was understandable that scientists had no idea where this technology would take them. Within a few years, this efficient, innovative system has found its way to the very heart of the field of gene editing; firmly establishing itself in efforts to find treatments for inherited diseases such as cystic fibrosis, and molecular targets within cancerous cells (Tian et al, 2019).
The mechanism of CRISPR, an abbreviation for clustered regularly interspersed short palindromic repeats, plays an important role in the immune reaction of type II bacteria against the exogenous nucleic acids of bacteriophages (bacteria-infecting viruses). The ‘spacer’ sequences of the CRISPR DNA region, containing information from previous viral invaders, are the ideal template for the creation of single-stranded RNA molecules which are complementary to the target sequence. Utilising the Cas9 nuclease enzyme, these RNA strands are able to introduce double-stranded breaks in the target genetic material (Song et al, 2016). In this way, infection of the host is effectively prevented. The recent application of CRISPR in human cells has turned it from an immune defence mechanism to a system capable of inserting deliberate mutations into genomes. Hence, it is clear that information can be manipulated. The next question that arises is whether this information can be permanently stored.
During their lifetime, cells are constantly exposed to a plethora of biological signals, from hormones and nutrients to enzymes responsible for adding or removing epigenetic marks and metabolism-inhibiting drugs. The pathway responsible for the transformation of a progenitor cell to its fully differentiated counterpart also relies on such biomolecules. An interesting theoretical implication of CRISPR/Cas9 technology is that these signals propagated into cells can be ‘written’ into the genome, in the form of gene edits, which can be stored over long periods of time. CRISPR-mediated Analogue Multi-event Recording Apparatus (CAMERA) was developed in 2018 and serves as an ideal early model to test the reaches of this theory (Fan, 2018).
The first model created (CAMERA 1) depended on the incorporation of two very similar plasmids (R1 and R2) into an S1030 E. coli bacterial cell. The plasmids used were only distinguishable from each other due to an alteration in three bases. The region containing different base pairs on the R1 plasmid acted as the target for DNA cleavage, by displaying complementarity to the nucleotides of the single guide RNA (sgRNA). After, only two additional promoters had to be added to create the CRISPR system: a TetO sequence upstream of the Cas9 gene, and a contingent Lac sequence upstream of the sgRNA. When the cells were treated with anhydrotetracycline (aTc), the TetO promoter was activated, and Cas9 and the sgRNA were subsequently transcribed. Thus, a double-strand break was introduced into the R1 plasmid upon exposure to the ‘trigger’. Plasmid replication functions in such a way that the downregulation of one will lead to upregulation of the other, and so the destruction of R1 led to an increase in R2 concentrations. Analysis of plasmid ratios confirmed this by showing an R1 percentage decreased from 58% to 4% over six hours. In another supplementary test, these ratio changes appeared to be stable in bacterial cells for the tested period of 144 hours (Tang and Liu, 2018). Combined, these experiments represent the fundamental research into CAMERA’s recording capacity.
Living organisms are, however, far more complex. Cells aren’t exposed to only one type of biomolecule in a single event, but rather to multiple, repeatedly and over a range of timescales. In order to mimic this, a system was developed whereby the Lac promoter of the aforementioned bacterial cell was swapped with a LacO promoter (which could only be activated by the molecule IPTG). No other changes to the CRISPR machinery were added, so the resulting cell would only require exposure to aTc to transcribe Cas9 and IPTG to transcribe the sgRNA. This was proved to hold, as only exposure to both molecules led to a significant reduction in R1 concentrations. Following on from this, another feature of CAMERA 1 developed on was its ability to reset plasmid ratios to their pre-exposure concentrations. In separate E. coli cultures, R3 and R4 plasmids were introduced into the bacterial cell. The sgRNA activated by the LacO promoter in response to IPTG, and responsible for cleaving R3, and was also included. In addition to this, another sgRNA needed for the destruction of R4 was incorporated. This RNA strand had the ability to be activated by a promoter in response to the biomolecule rhamnose. Therefore, alternating exposure to either IPTG or rhamnose led to a re-setting of R4 and R3, respectively, to their baseline levels (Tang and Liu, 2018). Such advances say a lot about the potential future applications of this extrinsic biological machinery.
Prokaryotic bacteria are used in a vast number of genetic engineering experiments, and this is greatly justified. Their relatively exposed, less complex circular chromosomes (in comparison to eukaryotic nuclear chromosomes) and lack of intracellular membranes make their DNA easy to manipulate (Magasanik, 1988). As a result, the real test of CRISPR technology is when it’s applied to eukaryotic cells. CAMERA is a good example of this, as an entirely difference mechanism (CAMERA 2) had to de developed to test its effects in human HEK 293T cells. In these cells, a third-generation base editor (BE3) protein complex, made up of a DNA base modification enzyme and a Cas9 nickase enzyme (capable of creating only single-stranded breaks), was put under the influence of a promoter which could only be activated by Wnt signalling. When lithium chloride (a molecule known to activate Wnt signalling) was added, BE3 and its dependent sgRNA were expressed. The sgRNA was able to guide the BE3 complex to a particular region of the CCR5 gene, and hence led to a C/G to T/A nucleotide change at the locus (Tang and Liu, 2018). Wnt signalling is just one of thousands of pathways active in a developing cell, and yet it is of great significance. For example, downregulation of Wnt signalling has been classified as an Alzheimer’s trigger, while upregulation has been linked to kidney disease (Ng et al, 2019). If the intensity of such a vital cascade can be recorded by a cell, what is the limit of CAMERA’s abilities?
Since the advent of CRISPR gene editing, the research world has been briming with ways to utilize it in healthcare. CAMERA is perhaps one of the leading technologies being investigated. However, only in vitro studies with cell cultures have been carried out so far. Will the same efficacy be observed in whole organisms? How will these CRISPR genes be introduced into entire tissues on a large-scale? There are still many issues that need to be addressed. While this is happening, multitudes of other signal recording technologies are also being developed, with acronyms such as GESTALT and SHERLOCK (McKenna et al, 2016; Gootenberg et al, 2018). We have a variety of tools to choose from. The only question remaining is how to use them in the most effective way to broaden scientific understanding.
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