“The scientific focus of our lab is to understand the mechanisms involved in the earliest stages of neurodegenerative disease in order to deliver therapeutic targets with the greatest potential to prevent dementia and Parkinson’s disease. We are based in the newly established UK Dementia Research Institute (UK DRI) at the University of Cambridge. UK DRI is a collaborative, inter-disciplinary Centre, combining world-leading expertise unique to Cambridge from chemistry, biophysics and structural biology with internationally leading research in the biology of neurodegeneration. The Metzakopian lab employs a variety of state of the art techniques and disease models towards implementing innovative experimental approaches that will help us test our hypotheses. These include, but are not limited to, CRISPR-Cas9 genetic screens and single-cell transcriptomics in human induced pluripotent stem cell derived neurons. By providing a stimulating, interactive, and cooperative research environment that supports independent thinking, our group provides an excellent platform for personal and professional development.”(Source)
The following has been condensed and paraphrased from an interview with Dr. Emmanouil Metzakopian on February 15th, 2019.
The analogy often used to describe CRISPR is that it is like a pair of molecular scissors, where does that analogy fall apart?
The protein Cas9 uses a guide RNA to find its target region, then through an enzymatic reaction it cleaves the DNA and causes a double stranded break. That cleaving action is more like a chisel that hits the DNA over and over again. It also causes mutations, though the cell is usually able to repair that damage.
How much is known about the evolutionary history of CRISPR?
CRISPR Cas9 comes from the adaptive immune system of prokaryotes. It is encoded in regions of bacterial DNA that we thought were junk DNA when first observed, then we saw that they encode guide RNAs that allow the bacteria to protect itself against viruses attempting to inject their genomic information into the bacteria, which would allow viruses to replicate and infect other bacteria. The bacteria has a memory of that event encoded in its immune system to protect it from future viral infections. It took some very intelligent people to observe this process and figure out that it could be applied to mammalian cells as well.
You’ve build a very extensive CRISPR library, what is a ‘CRISPR library’?
The Cas9 protein targets the genome using a guide RNA sequence that is about 100 bases long. The part that is used for targeting is 20 bases long and can be changed to target any part of the genome. You can design as many of these sequences as you’d like to target virtually as many genes as possible. Sets of these sequences are called a library. It is a physical sequence of plasmids we keep on a shelf that can be used to target any gene of interest. They are also easily replicable and should last forever.
Which applications of this library are you most excited about?
The main application we use it for is for genetic screening purposes. We use them on dopaminergic cell lines to find genes that would change the phenotype in a positive way to see which genes and which pathways might have therapeutic value.
What are the upper limits of what CRISPR will allow us to do?
CRISPR is speeding up discoveries at a rate we have never seen before. In neurodegeneration very soon we will surpass the bottleneck of identifying new mechanistic insights in neurons thanks to CRISPR. Coupled with iPS derived neurons we should have some amazing discoveries coming in the near future. At a minimum this will result in many more therapies being tested in the clinic within the next 10 years. We might also use it in gene therapy to cut genes that we know are causing harm. But we are still learning how CRISPR works so I wouldn’t put it in humans yet because it does have off-target effects and the mutations that it causes are not very well controlled. But new proteins are being identified that are safer and will help improve this technology.
Could you conceive of any other tools on the way that might cause a similar paradigm shift?
10 years ago if you had told me that a protein can be identified to mutate any gene of interest at the push of a button I would have said that sounds like something out of Star Trek. Now there are other such tools that are having a huge effect, such as the new use of peptides called degrons that allow us to degrade any protein of interest. That is the next step, not just destroying genes but manipulating their expression. I hope these tools will continue to expand and evolve and soon will be used for personalized medicine, that’s the dream I would like to see.
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