Jun 25 2021


Bonus points for anyone who has managed to commit to memory what the CRISPR acronym stands for – Clustered Regularly Interspaced Short Palindromic Repeats. I still have to look it up each time to make sure I get it right, but I’m getting there. I first wrote about CRISPR in 2015. It is a method of editing genes derived from bacteria. CRISPR itself is a means of targeting a specific sequence of DNA; you load it with the desired gene sequence and it will find the corresponding sequence in the DNA. Of course, it has to do something once it gets there, so CRISPR is also combined with a payload, such as Cas9, which are molecular scissors that will cut the DNA a the targeted location.

Since the potential for the CRISPR-Cas9 system in genetic engineering was realized, the technology has been on a steep climb of advancement. This was a new platform, one that had the advantage of being relatively quick, easy, and cheap. This means that genetics researchers round the world were all able to play with their new toy, and not only find uses for it but find ways to improve it. The basic technology slices DNA at a desired location. One limitation of this technology, as accurate as it is, there are still off-target effects. But researcher have already started to unpack how to make CRISPR slower but more accurate (vs faster but less accurate). They know how to dial in the accuracy, and it’s likely this aspect of the technology will improve further.

Also, once you make a slice in the DNA this can have a couple of effects. If all you want to do is kill the cell (like a cancer cell) you just make a bunch of slices and be done with it. If you want to inactivate a single gene your job may also be done. However, if you want to edit the gene then there is another step. You need to coax the cell into using its own repair mechanism to fix the break in the DNA while simultaneously inserting a new bit of DNA into the break. This is full gene editing, and while it’s still a bit tricky, it is much faster and cheaper than other methods.

Cas9, however, is not the only payload you can pair with CRISPR. Part of the research is looking at other version of Cas that have different functions. Earlier this year, for example,  I wrote about CRISPR On-Off.  This uses a “single dead Cas9 fusion protein” in order to turn a targeted gene off. This does not alter the gene in any way, except to turn off its expression. Because the gene itself remains intact, you can then turn it on again.

With CRISPR we can edit genes, silence genes, destroy DNA to kill specific cell types, and can turn genes on and off. Researchers have recently improved upon another potential application of CRISPR – gene amplification (increasing the expression of the gene, or how much protein it makes). This is the CRISPR-Act technology, and the authors are calling the new version CRISPR-Act3.0. This uses a deactivated Cas9 payload in order to recruit transcription activators. Transcription is the process of making a messenger RNA (mRNA) from a gene in the DNA. The mRNA is then used to make a specific protein. Transcription activators, therefore, increase the rate of transcription and the amount of protein that’s made.

The improvements in 3.0 include greater efficiency, a 4-6 fold increase in the amount of activation over older versions, and that ability to target up to 7 genes simultaneously. I should point out this research is being done entirely in plants. What practical uses does gene amplification have?

For genetics research, this is incredibly useful. If you want to find out what a gene does, amplify the crap out of it and see what effect it has on the cell or organism. This would complement the “knockout” approach, which researchers can now do much easier with CRISPR-Off. So genetics labs can now affordably turn genes off and on and amplify them to see what effects it has. This will accelerate the pace of genetics research.

For translational applications (so with practical effects) this can be used to quickly adapt plant varieties to different environmental conditions. Evolution does not always require new mutations or genes. Many complex organisms already have lots of genes for lots of things, and adaptation is a matter of turning up or down the expression of those genes. In fact, this is largely what epigenetic changes are all about – altering gene expression in response to environmental conditions. Epigenetic changes, however, are temporary, even if they can last for multiple generations. They are short term tweaks to make plants and animals more adaptable to current conditions.

CRISPR-Act gives us direct control of this one aspect of gene expression, so if we want to adapt a variety of rice to drier conditions, we just amplify genes that confer drought resistance, for example. We may want plants to flower more, grow faster, or have greater resistance to a specific pest. We don’t need to edit or introduce new genes, we can just amplify what is already there. Obviously there will be limits to this approach. We can only amplify what is already there. But still there is enormous potential.

When I first heard about CRISPR I knew this was going to be a technology to watch. In the past 6 years it has exceeded even my technophile optimistic expectations.

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