This post was written by Kirsty Oswald, Freelance Science Writer
The potential and pitfalls of CRISPRa
Many people are familiar with CRISPR-Cas technology as a genome-editing tool that acts like “genetic scissors,” allowing scientists to remove and insert sections of DNA into a cell’s genome. But when looking to apply CRISPR-Cas therapeutically, modifying genetic transcription, rather than editing DNA, could offer some advantages.
In a recent article (https://link.springer.com/article/10.1007/s00018-022-04175-8) in Cellular and Molecular Life Sciences, Elvir Becirovic from the University of Munich explores the progress and challenges of using gene-activating CRISPR-Cas modules (CRISPRa) in a therapeutic setting.
What is CRISPRa?
Just as in gene-editing, CRISPRa uses the Cas9 protein but without its endonuclease, the enzyme that allows it to cut DNA. Instead, it is coupled to other domains capable of transcriptional activation, such as by recruiting transcription factors. This CRISPRa package is then carried into the target cells with the help of a vector. The result is increased expression of the target gene.
So far, in a pre-clinical setting, CRISPRa has been successfully applied to a number of genetic or acquired diseases including epilepsy, muscular dystrophy, retinitis pigmentosa and cancer.
The advantages of CRISPRa-based approaches
One of the features that makes CRISPRa particularly suited to therapeutic applications is that it generates little to no off-target effects in vivo; it is a highly specific approach. And as CRISPRa targets regulation of transcription, it can be used independent of target gene size. For the same reason, unlike CRISPR-Cas approaches to remove or correct a disease-causing mutation, it is independent of mutations and therefore could be applicable to a wider number of patients. And, unlike genome-editing approaches, CRISPRa does not generally induce permanent changes to the genome.
The disadvantages of CRISPRa-based approaches
To date, CRISPRa research with a translational focus has almost exclusively used adeno-like virus (AAV) vectors. While much of this research has been successful, this approach to delivery is also one of the main limitations for applying the technology therapeutically.
Compared with other potential vectors, AAV vectors are particularly well suited to achieving long-term changes in gene expression and also have low immunogenicity. But, one key limitation is their DNA uptake capacity, which means they are not suited to carrying large cassettes.
What’s more, the aim of CRISPRa is to maintain permanent expression of the gene-activating module in target cells, which makes in unsuited to some alternative delivery methods, like liposomes, virus-like particles, and nanoparticles. So, the technology requires the development of new delivery methods for efficient and long-lasting expression of CRISPRa cassettes in vivo.
Other factors that could limit the use of AAV vectors in clinical trials include the pre-existing immunity to the virus in human populations, as well as the currently high cost of manufacturing. However, this cost may come down in future as production and competition increases.
Looking to the future
Despite the disadvantages discussed above, CRISPRa remains a promising tool that could one day be used in a clinical setting. As Becirovic concludes, “…although some barriers to gene delivery still need to be overcome and the immunogenicity better investigated, CRISPRa holds great translational potential and its implementation into first clinical trials could benefit millions of patients worldwide.”
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