Not every genetic modification has to involve a violent cleaving of nucleotides to insert or remove sections of the genome. The most common form this type of change takes involves what are termed double-strand breaks (DSBs) and they have long been something that scientists have wanted to avoid if possible. You may remember us discussing a similar topic involving DSBs previously. But the available tools for gene editing often did not allow for alternative methods of manipulation that didn’t require cleavage. Several years back though, a CRISPR researcher found a solution.

Raising The Dead

Back in 2013, one of the earlier papers that Jennifer Doudna and others published on CRISPR involved an alteration of its properties. Termed dCas9, this CRISPR system was made to be catalytically-dead, meaning that it lacked the nuclease activity the original system has and thus can no longer cleave DNA or RNA. This new usage was put to task with the same job it had had previously, but since it could no longer change the genome, they added a different capability by fusing the dCas9 with a transcriptional activator.

What this meant is that the dCas9 could then be guided to the target gene by the same guide RNAs (sgRNA) as usual and the transcriptional activator component fused to it could be put to work upregulating and overexpressing the gene in question. The only issue with this early experiment was that the amount of expression obtained was far too low to be useful for any therapeutic treatments. Doudna and company then went on to other CRISPR experiments, leading to the big gene editing reveal in 2015 that you all know about.

Later scientists attempted to fuse multiple transcriptional activator domains to the dCas9 in order to boost this efficiency and they were successful at target gene activation in in vitro (in cells in a lab) studies. But their results in vivo (in living, often multicellular, organisms) were less than adequate. The dCas9 fusion was just unable to properly target the needed gene once it was injected into a complex organism. Tied to this issue was that their complex fusion had become too large for the more common transport systems, such as adeno-associated viruses (AAVs), to be able to fit them.

Upgrading the Dead

Therefore, this year, researchers at the Salk Institute decided to see if they could figure out a better way to use dCas9. Their first goal was to see if they could design a system that didn’t require a bulky fusion system in order to function.

First, they chose to use short guide RNAs that, in their brevity, also lack the ability to activate Cas9’s cleaving response, resulting in them being called dead sgRNAs (dgRNAs). Of course, with dCas9, cleavage capability isn’t there in the first place, meaning the dgRNAs can be used and will fit in a much smaller package. They were also modified to have high expression at the target gene. The shortened guides made the whole group just small enough to fit them and the fusion dCas9 into an AAV transport.

Other combinations of transcription activator complexes were tested, with one promising note being that optimized dgRNAs allowed high levels of target gene activation even without the complexes being included at all. After a series of experiments showing that their system was able to properly activate genes in adult mice, they wanted to demonstrate the therapeutic applications of this modified Cas9 setup.

Taking individual sets of mice with a range of disorders, specifically acute kidney injury, type 1 diabetes, and Duchenne muscular dystrophy, they applied their system to overexpress genes that run counter to the effects of the condition. For example, for the mice with muscular dystrophy, they targeted genes involved in muscle growth, whose expression would offset the negative symptoms of the disorder. All of these were found to be completely successful at reducing the disease symptoms and improving the quality of life for the mice.

A final experiment they ran involved splitting the dCas9 from the dgRNAs and transcription activators and placing each into optimized AAV transports. They found that co-injecting these showed the best target gene activation efficiency, where expression levels were multi-fold above their prior tests.

Transient Medicine

Overall, this modernized and updated dCas9 structure should allow medical researchers to therapeutically counteract the symptoms of many genetic disorders. If combined with other methods that also try to correct the mutations, it may be possible to completely halt the onset of such disorders in adults, if enough efficiency in expression can be achieved.

Sure, this option treating the disorders may only be transient and potentially require monthly injections to keep the effects going, but it is a grand step forward in our treatment of some of the most intractable genetic diseases where, just a few years ago, we had little to no recourse in helping those suffering from them.

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Photo CCs: Compact bone – ground cross section from Wikimedia Commons