CRISPR-Cas9 is one of the hottest topics in molecular biology (see this Nature special), and with recent advances is starting to gain attention in the mainstream media; see NYT, Guardian, BBC and many, many more news sources.
A lot of this interest has been driven by the case of Brian Madeux in California. Brian, who has the rare metabolic disorder Hunter syndrome, (which is caused by mutations in the IDS gene leading to lack of activity) became the first person to receive a treatment based on specific genome editing. In this case the therapy was based on a similar technology called zinc finger nucleases, but the focus is on CRISPR-Cas9 as this is now thought to be a superior, albeit newer, technology.
In this post I’m going to give an overview of genome editing by CRISPR-Cas9 and the other technologies as well. But I realize some of you might not get quite as excited by the technical details as I do, so I’m going to do it in two parts. A super simple primer below, followed by more technical details for those interested.
Genome editing: Super-simple summary
A major issue surrounding genetics, life science and research as a whole is how to communicate the jargon filled areas of interest to an audience who might be interested, but don’t have the background understanding to get all the acronyms.
Metaphors are great to use in this instance and I always remember the example a lecturer from my undergraduate years used when discussing science communication. To her, enzymes became “molecular scissors or molecular glue” — they either cut other molecules apart, or helped stick them together.
Simple, easy to understand and applicable to many technologies. And so as a metaphor you would often see it used to discuss genome editing. Scientists could use enzymes to “cut” our genome to either delete, insert or replace bits of our genetic code and then glue it back together again.
But with previous genome editing technologies this metaphor implied a level of accuracy that just didn’t exist. When you use a pair of scissors, you line them up carefully and make a cut exactly where you want. But with techniques like viral gene editing, which was/is a mainstay of life science research this accuracy simply wasn’t there. Tools like this (and others) could insert, delete and modify DNA but our control about where this happened was very limited.
Why does this lack of control matter? Well two reasons, firstly when targeting a specific region such a lack of accuracy means researchers would need to make many attempts to get the desired effect. The second, and much more profound issue (especially when thinking of using these tools in the clinic) is the fact that randomly inserting DNA into the genome of a living cell, can, lead to that cell becoming tumorgenic, i.e. cancer causing. I’ve bolded can as the chances of this happening are very low, but they do occur. See this example of a child with a severe disorder of the immune system who developed leukemia following gene therapy treatment.
So I’d like to alter that metaphor and say such techniques were like using “molecular scissors and glue” but whilst wearing a blindfold. You will have an effect, but it may not be the one you wanted.
Enter CRISPR-Cas9, TALENs and ZFNs. I won’t bore you with the full names in this bit, but recently developed and developing technologies are an evolution on what has gone before. The mechanisms all differ slightly but what these technologies do, is allow accurate targeting (in the case of CRISPR-Cas9 we’re talking incredible accuracy) of specific regions of the genome. So if the previous generation of technologies were using “molecular scissors and glue” with a blindfold, these technologies very definitely mean the blindfold has come off!
What does this mean for us? Well the first uses of the technology are going to focus on severe disorders and diseases in the way all these technologies do, due to cost and complexity. But within a few years this technology will be commercially viable. So say you carry the risk allele ‘T’ for A16V (T47C) in the SOD2 gene. We know this allele is associated with an impaired antioxidant profile and is associated with several disorders such as diabetes and certain cancers. Well using CRISPR-Cas9 researchers could target this region of the SOD2 gene and snip out that risk ‘T’ and replace it with a healthy ‘C’, bringing your antioxidant profile back up to health levels.
There are some issues to overcome, namely exactly how the therapy would be delivered throughout the body. Certain tissues are much easier to target then others, for example our bone marrow, which provides the cells of our immune system is relatively easy to target, but other solid organs like the brain or heart are much more difficult. Additionally, what happens next will depend a lot on regulation, but a future where you can pop into the clinic and have a harmful SNP “fixed” doesn’t seem that far off.
I hope that makes sense as to why people are so excited about these technologies, and if you’re interested in getting some more technical understanding I’ll go into that below.
Genome editing in depth
I’m going to use science score a little differently here and rate how likely the technology described is to become widely used in the clinic and then also by researchers.
Zinc finger nucleases
Zinc fingers are small regions of proteins characterised by a fold in the protein which is stabilized by one or more zinc ions. These folds allow for short specific regions of a protein to be exposed away from the rest of the protein allowing for highly specific interactions with other proteins or DNA and RNA molecules.
Importantly, there are many types of zinc fingers which interact with different DNA coding regions, for example one might bind with an AAT sequence, while another may bind with a CGC sequence.
Researchers used this high level of DNA specificity to make the first type of precision gene editing tool, the zinc finger nuclease (ZFN). ZFNs typically contain up to 6 individual zinc fingers and by carefully combining these zinc fingers researchers were able to make enzymes that could target specific regions of DNA with a high level of accuracy.
By tagging a nuclease enzyme (which cuts DNA) to these constructs researchers created a system where they could cut DNA with much more specificity than was previously possible, allowing for targeted deletion or insertion of any gene, or region of a gene. Theoretically allowing for the correction of SNPs.
Image from Carroll, D. Genome engineering with zinc-finger nucleases. Genetics Society of America, 2011, 188(4), pp 773-782.
Downsides of ZFN
Unfortunately, things aren’t quite as rosy as the diagram above would lead you to believe. Many zinc fingers altered their specificity based on other zinc fingers around them. So a zinc finger might target CGC on its own, but when next to another different zinc finger it might target CAA. Making these molecules and then testing their specificity is a huge task and so the search was on for an improved method.
Enter transcription activator-like effector nucleases (TALEN). In a similar fashion to ZFN TALEN relies on combining a protein capable of targeting highly specific regions of DNA with a nuclease enzyme, which can cut DNA.
In this case DNA is recognized by proteins known as TAL effectors which are isolated from a particular bacteria. Rather than using multiple zinc fingers, only a single TAL effector is required, so issues with specificity (the major shortfall of ZFN) are bypassed.
Downsides of TALEN
The specificity of TALENs are much greater than that of ZFNs. However, there are issues with the constriction of TALENs in the lab, being protein based they are more complex to make and a new protein must be created for each individual DNA region to be targeted. So while a very accurate technique it remains expensive and relatively time consuming.
Finally onto the main subject of the post. CRISPR-Cas9 is similar to both TALEN and ZFN in that it allows precise genome editing, the difference is in the mechanism of action. Both TALEN and ZFN combine a protein(s) which recognises a region of DNA with a nuclease which then cuts the DNA at that point. Whereas CRISPR-Cas9 combines a piece of RNA with a protein nuclease.
This region of RNA is used to target the specific region of DNA to be targeted and making RNA in the lab is much quicker, cheaper and also allows for a much higher degree of accuracy than either other method.
CRISPR in this case refers to the RNA sequence and Cas9 is the nuclease protein which again is isolated from bacteria. In the image below you can see a diagrammatic representation of this with the grey DNA being targeted by the green RNA, which is then cut by the blue protein Cas9.
Image from Wikipedia.
So to summarize. Scientists now have access to molecular tools which allow them to manipulate the genome with a high degree of accuracy and with relative ease. However, there are still several hurdles that need to be overcome, firstly a major issue with all these technologies is about how to deliver them safely to wherever they need to go. It’s to be able to fix a gene relating to a severe neurological disorder, but how do you get that into a person’s brain? Safety is also key. While scientists are predicting much fewer side effects from these technologies, there are still risks.
As they are all so new at the clinical level, we have no idea how things will perform. Expect to see many more clinical trials occurring and we’ll report on any findings as and when they come out. The first trials are focused on using ZFN, as this technology has had longer to mature, but expect to see work dealing with TALENs and perhaps most excitingly CRISPR-Cas9 to start hitting the news soon!