Credit: MIT News

It may be possible to accurately modify gene transcription in vivo using CRISPR without cleaving DNA, according to recent research. Previous attempts at using Cas9 enzymes to modify transcription have been restricted by the size limitations of delivery vectors; a different approach has been developed which may be able to avoid these issues. A review of the work was published in Nature Reviews Genetics today.

The CRISPR-Cas9 system has significant potential as a treatment for genetic diseases, as it can move to the precise genomic locus and cleave the DNA double strand. Despite its accuracy, however, CRISPR does carry the potential for ‘off-target effects’ which involve genes that are not being targeted becoming mutated. In vivo, this could have significant and unpredictable side effects and this has meant that the system needs to be improved before it can be used in the clinic.

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One potential solution is to use a variant of the traditional Cas9 nuclease called dead Cas9 (dCas9), which has lost its nuclease activity. Instead, a guide RNA (gRNA) leads the protein to the correct genomic locus, where it binds without causing a break in the double strand. If the dCas9 is fused to a protein domain capable of influencing transcription (such as an activator or repressor) then the complex is capable of performing targeted transcription modification.

Unfortunately, delivering the dCas9 fusion protein into cells has been difficult. With traditional CRISPR experiments, it is possible to insert the source DNA code into cells via an Adeno-Associated Virus (AAV) vector. The problem with this approach is that AAV vectors are strictly size-limited, and the DNA code for the dCas9 fusion proteins are very large. As a result, the vectors cannot be used.

Recent research, originally published in Cell, offers an alternative approach. The team adapted the gRNA molecule that is used to guide Cas9 so that it could independently recruit the engineered transcriptional activator MPH. The gRNA was also truncated so that even when it forms a complex with fully functional, wild-type Cas9, it is unable to form DNA breaks; as a result, the team called it nuclease dead gRNA (dgRNA).

They were able to demonstrate that dgRNA and MPH could be contained within a single AAV vector, while a second vector could be used to transmit the DNA code for Cas9. The team then tested this combination against a range of genetic diseases in mouse models, with wide spread success (many of the mouse models were transgenic and expressed Cas9 prior to the experiment). Importantly, the research demonstrated up-regulation of large genes, such as utrophin, which have been difficult to influence in the past because of their size.

“Overall, this system has numerous attractive features as a potential human therapeutic for various disorders, particularly the lack of overt genome modification by either Cas9 or the non-integrating AAV vector,” the review concludes. “However, with such properties it will be important to determine whether therapeutic benefit can be achieved long term, such as how long transcriptional modulation can be sustained after a single injection, and whether the potency of serial injections will diminish through immune responses to AAV or its payloads.”

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