SITN_CRISPR_Neuroscience_cover

by Angela She
figures by Shannon McArdel

The brain is one of the most complex entities in biology. For thousands of years, humans have wondered how the human brain works, but only in the past few years has technology evolved so that scientists can actually answer some of the many questions we have. What are the causes of brain disorders? How do our brains develop? How does the brain heal after a head injury? While we still have a long way to go before we can understand the many facets of the human brain, one technology – CRISPR – has allowed us to start answering these questions on a genetic level.

What is CRISPR?

CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, is the latest in a long line of genome editing techniques. Over the past few years, CRISPR has swept through the biology community and into mainstream news. The technology, which can be used to make specific changes in the DNA of plants and animals, has become instrumental to studying disease systems in the lab because of its low cost, precision, and ease of use. Unlike other genome editing methods, scientists can use it to change any stretch of DNA in a genome, as long as they know the sequence to target. They can make multiple changes in one fell swoop.

The CRISPR system has two parts: 1) a protein borrowed from bacteria that cuts DNA, and 2) a guide RNA that tells the protein where to cut. Once the DNA of the gene being targeted is cut, the cell will try to repair the DNA but will often make a mistake, causing the gene to be disrupted and no longer function. Sometimes, scientists will also introduce a third component of the system: a DNA template that tells the cell how to repair the cut DNA and introduce a very specific mutation that changes the gene in some way. Either way, scientists can use CRISPR to alter the DNA inside cells (Figure 1). (Learn more specifics about CRISPR and how CRISPR works here.)

 

Figure 1: How CRISPR works. A guide RNA that matches the genomic DNA sequence of interest helps direct a CRISPR protein toward this site in the DNA. The protein cuts the strands of DNA. The cell will try to repair this break, but during this process may introduce random mutations that render the gene non-functional – the gene is thusly silenced. However, if scientists introduce a DNA template that is slightly different from the original DNA sequence, the cell can use it to guide the repair and introduce a specific mutation into the gene sequence.
Figure 1: How CRISPR works. A guide RNA that matches the genomic DNA sequence of interest helps direct a CRISPR protein toward this site in the DNA. The protein cuts the strands of DNA. The cell will try to repair this break, but during this process may introduce random mutations that render the gene non-functional – the gene is thus silenced. However, if scientists introduce a DNA template that is slightly different from the original DNA sequence, the cell can use it to guide the repair and introduce a specific mutation into the gene sequence.

Why is CRISPR So Important for Neuroscience?

The advent of CRISPR has been timed perfectly with the recent neuroscience research boom. Over the past few years, scientists have been using gene sequencing to uncover genes that are important in brain development and in neurological diseases, like Alzheimer’s disease and schizophrenia. Whereas some neurodevelopmental disorders, like Fragile X Syndrome, are known to be caused by mutations in a single gene, diseases like schizophrenia involve many genes and are very complicated, so thousands of people had to be sequenced before scientists could figure out which genetic differences might be linked to the disease. Today, there are clues to the genes that might affect a number of disorders, like OCD, autism, and major depression. The next step is to figure out if disrupting these genes can cause any of these diseases. CRISPR seems to be the perfect technology to make this happen.

Because the brain is the product of millions of connections between neurons, it’s important to see what these genetic changes do in an actual animal brain. If we think that a specific mutation in the Huntingtin gene causes Huntington’s disease, we can introduce that mutation into the embryo of a mouse via the CRISPR system. These mice and their offspring will contain this mutation and we can study their behavior and physical changes and see if they have the “mouse version” of Huntington’s disease. These mice can then be given potential drugs to see if those drugs help relieve symptoms (Figure 2).

Figure 2: Using CRISPR to study neurological diseases in model organisms. Once a possible genetic link has been found to a neurological disease like Huntington's disease, Alzheimer's disease, or Parkinson's disease, scientists can use the CRISPR system to introduce the relevant genetic mutations into model organisms like mice. By understanding the differences between mice with the genetic mutation and mice without it, scientists can paint a clearer picture of how the disease might be affecting a human body. Then, the mice can be given potential drugs or treatments that can help to alleviate their symptoms or even help cure the disease.
Figure 2: Using CRISPR to study neurological diseases in model organisms. Once a possible genetic link has been found to a neurological disease like Huntington’s disease, Alzheimer’s disease, or Parkinson’s disease, scientists can use the CRISPR system to introduce the relevant genetic mutations into model organisms like mice. By understanding the differences between mice with the genetic mutation and mice without it, scientists can paint a clearer picture of how the disease might be affecting a human body. Then, the mice can be given potential drugs or treatments that can help to alleviate their symptoms or even help cure the disease.

Previous methods for making mouse models can take up to two years from design of the mutated gene to multiple rounds of mouse breeding to make sure that the mouse offspring have the correct genetic mutation. In contrast, it takes only about two months to create a mouse model using CRISPR because the components are more easily introduced into the embryo and multiple breeding steps are not required. In addition, if we think that more than one gene is contributing to a disease like schizophrenia, we can easily introduce two or more mutations into a mouse embryo at the same time simply by introducing multiple guide RNAs (and template DNAs), something that is not easy to do with other genome editing technologies (Figure 3).

Figure 3: Timeline of CRISPR compared to a traditional genome editing technique. For diseases like OCD that are known to be caused by more than one genetic mutation, making model organisms with multiple genetic mutations is crucial to understanding the disease. With traditional genome editing methods, it might take up to three years to create a mouse model with two genetic mutations, with CRISPR, scientists can create a mouse model with one, two, or even more genetic mutations in as little as six weeks!
Figure 3: Timeline of CRISPR compared to a traditional genome editing technique. For diseases like OCD that are known to be caused by more than one genetic mutation, making model organisms with multiple genetic mutations is crucial to understanding the disease. With traditional genome editing methods, it might take up to three years to create a mouse model with two genetic mutations, with CRISPR, scientists can create a mouse model with one, two, or even more genetic mutations in as little as six weeks!

With CRISPR, dozens of mouse models and other animal models have been made to study neuroscience. For example, the Zhang lab at the Broad Institute in Cambridge, MA have used CRISPR to make mouse models of OCD and autism. Mice with OCD-related genetic mutations groom themselves more and seem to be anxious about their environmental cleanliness and mice with autism-related genetic mutations are generally less sociable than other mice. The Zhang lab is currently working on making mouse models with particular mutations in a gene called Shank3 which might be important in both autism and schizophrenia.

The Challenges of CRISPR

CRISPR is still a relatively new technology and it’s not perfect. The human genome is large and sometimes, multiple stretches of DNA are similar enough that the CRISPR system will make unintended cuts in the DNA. In this way, unintended mutations might arise which might affect the health or even survival of the animal and can confuse the results of any experiments. Many researchers are currently studying ways of making the CRISPR system more specific so that only the genes one intends to target are affected.

Right now, it is easy to inject CRISPR components into mouse embryos, but if scientists want to introduce CRISPR into an adult rat brain (perhaps with therapeutic intent), they’re out of luck. It is very difficult to get the CRISPR components to cross the blood-brain barrier. Some progress has been made by stripping the CRISPR components down and stuffing them into a modified non-disease-causing virus that can easily cross the blood-brain barrier. Think of this like organizing your carry-on bag (the virus) with only your essentials (the CRISPR components) such that it just makes the size cutoff at the airport. However, although the virus package has no disease-causing power, the long term effects of using such a virus and the ramifications of stripping down the CRISPR components on their effectiveness in the brain are still being investigated.

Finally, there are ethical issues to consider when proposing CRISPR as a gene therapy for humans or even using CRISPR in primate animal models. While primates are used in a number of brain imaging studies, the ethics of genetic manipulation in these animals, and potentially in humans, is still being hotly contested.

The Future of CRISPR

Even as research is being done to improve the specificity and efficiency of CRISPR itself, neuroscientists are finding more inventive ways to use the technique, developing model organisms not previously used in neuroscience, such as social insects like locusts, more sophisticated social mammals like bats. Many neurological disorders cause people to behave unusually in social situations, and studying other social animals might help us to understand why that is. Many scientists are also using CRISPR in human induced pluripotent stem cells (see this special edition article) or in neurons derived from these stem cells to study the effects of genetic changes on human neurons in a dish. As neuroscientists create more animal and cell models with CRISPR, we will be able to unravel more about what makes the brain tick and how to fix it if it breaks.

Angela She is a PhD student studying neurodegenerative diseases at Harvard. She is also one of the co-producers of the SITN podcast, SIT’N Listen.

This article is part of the April 2016 Special Edition on Neurotechnology.

Further Reading:

Featured image from AstraZeneca.

To read more about the development of the CRISPR genome editing system and the ethics surrounding it: Read here and here

To learn more about CRISPR crossing the Blood-Brain Barrier: Read here

To learn more about CRISPR and specific genetic mutations, like Shank3: Read here

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