Читать книгу Genome Editing in Drug Discovery - Группа авторов - Страница 51

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.