fusing a domain of Adenosine deaminases acting on RNA (ADAR), key enzymes involved in RNA editing, allows one to post‐transcriptionally change the sequence of RNA from adenine to inosine (decoded during the translation as guanine), altering the protein primary sequence without affecting the genome (Cox et al. 2017) (Figure 3.7g).
Overall, the adaptation of RNA‐targeting type VI CRISPR systems now allows researchers to manipulate the transcriptome, a powerful and complementary tool to manipulating the genome by other systems.
3.4.2.3 Biochemical Detection
Finally, we would like to point that CRISPR systems can be used for molecular diagnostics. The collateral activity of Cas13 proteins, while conceptually undesirable, has been exploited to develop highly sensitive detectors for the presence of specific RNA. This approach relies on in vitro activation of Cas13’s indiscriminate RNase activity by an on‐target recognition of specific RNA. Once activated, Cas13 is able to degrade other RNA molecules in the reaction mix; if a fluorescent RNA reporter molecule is included, then Cas13 is able to detect a specific RNA molecule with attomolar sensitivity (Gootenberg et al. 2017), and improvements claiming to bring sensitivity down to zeptomolar (10−21 mol/l) range (Gootenberg et al. 2018) (Figure 3.7h). This system, termed SHERLOCK, has been used since to develop fast and sensitive methods for diagnostics of a number of highly pathogenic RNA viruses (Myhrvold et al. 2018; Patchsung et al. 2020). A similar approach exploits a collateral activity of Cas12a proteins on ssDNA, allowing one to detect specific DNA (or cDNA) sequences; indeed a flurry of different variants of this concept have been developed to detect pathogen DNA or cDNA or to perform SNP profiling (Chen et al. 2018; Li et al. 2018b; Teng et al. 2019b). These developments and innovation are momentous and provide a major progress for molecular diagnostics (Li et al. 2019b).
3.5 Concluding Remarks
The discoveries made in the field of CRISPR biology in the last two decades have paved a way for efficient, cost‐effective, and precise genome editing. Efforts in biochemical and structural characterization of a great number of Cas proteins expanded the toolset of CRISPR proteins one can use for genome manipulation and other purposes. Initially, SpyCas9 was the only Cas protein used for genome editing, but the development of a multiple Cas9 variants with expanded PAM targeting landscape, the discovery of Cas9 orthologs and finally characterization of new class 2 proteins, has in a manner removed constraints imposed by the biochemistry of Cas9. Thanks to an ever‐expanding collections of available Cas nucleases, in the years to come we can expect to perform gene editing with the best tool for a given sequence and application, and without compromising precision and efficiency. Indeed, one can draw a parallel between the democratization of restriction enzymes for molecular cloning and CRISPR enzymes for genome editing, where we are now able to use a bespoke enzyme (rather than relying on a modest set) for diverse and complex outcomes.
Many innovative methods have been developed to mitigate pitfalls of these novel technologies: low efficiency can be enhanced by fusing the Cas proteins to more efficient nucleases (Dolan et al. 2019); specificity can be improved by careful crRNA design (Doench et al. 2016; Akcakaya et al. 2018) and enhanced by blocking unspecific sites (Coelho et al. 2020), the activity can be controlled by chemical agents (Maji et al. 2017) and optogenetically (Nihongaki et al. 2015). Whereas these tools are a powerful addition to the CRISPR arsenal, recently discovered inhibitors of CRISPR systems, the anti‐CRISPR proteins (Acr) provide the basis for even tighter control of undesired activities of Cas proteins (Davidson et al. 2020; Marino et al. 2020). Acrs are, in essence, an evolutionary response of mobile genetic elements able to inhibit CRISPR systems at various steps of the immune response. Just like CRISPR systems, their inhibitors are incredibly diverse, and thus represent yet another untapped resource of wonderful tools that can be used to further improve CRISPR editing, in particular in a therapeutic setting.
Studying the biology of diverse CRISPR systems did not lead just to the development of gene editing tools, but also ways to manipulate the transcriptome and the epigenome. Furthermore, by exploiting the seemingly undesirable collateral activity of type V and type VI systems, new molecular diagnostics tools with unprecedented detection sensitivity have been developed. Together, this diverse collection of novel ways to repurpose bacterial immune systems for a bespoke application is a witness of how much more we can get by understanding and studying microbial CRISPR systems. While class 2 systems have been studied (and hence appropriated for various applications) to a great extent, the application of class 1 systems is lagging. Furthermore, other phases of CRISPR immune response are still comparably poorly characterized, for example, adaptation phases. Studying these systems and these phases might well generate new powerful tools for genome editing or something completely different. One can only eagerly wait for what the next decades are going to bring.
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