CRISPR Fundamentals

Let’s walk through some of the CRISPR basics. The term CRISPR is an acronym for:

Clustered

Regularly

Interspaced

Short

Palindromic

Repeats

There are many proteins involved and they are all part of a family of proteins that we call Cas proteins (Cas is short for CRISPR-Associated).

There are two primary components to CRISPR: A Cas protein that interacts with (takes action/does the work on) DNA, and a small piece of “guide” RNA that helps the Cas find its target DNA sequence.

This probably doesn’t clear anything up and it doesn’t really sound like it has anything to do with gene editing.  That’s because CRISPR was initially discovered as a naturally occurring phenomenon in bacteria and later manipulated into a tool that scientists can use.

The natural CRISPR mechanism in bacteria forms a simple but elegant way for the bacterial cell to defend against invading viruses: Bacteria have short DNA sequences that match different viral DNA sequences, so they stick together like velcro. These DNA sequences are made into “guide RNA”, which the Cas protein uses to velcro onto the DNA of invading viruses. The Cas protein then cuts the DNA like a pair of scissors, inactivating the virus.

How is CRISPR used?

Already, you may be able to see how this system can be leveraged by genetic researchers; if we target human DNA instead of viruses, we can change the expression of specific genes and their proteins.  The star of CRISPR is the guide RNA: For DNA editing, specificity is a top priority to avoid unintended side effects.  Researchers can target specific genes simply by changing the recognition sequence of the guide RNA. There are some limitations to this, but it is still an extremely versatile system.

So let’s talk about strategies for using CRISPR to edit the human genome.  Due to its versatility, CRISPR can be used in many different aspects of research and medicine.  Before we can attempt to fix a broken gene, basic research needs to establish exactly what is broken, how it affects different cells, and the mechanism by which the genetic defect leads to patient symptoms.

I will go over two of the basic ways CRISPR is used in research.  For each of these examples, let’s imagine that we have a petri dish of human cells that we are using to study SLC13A5.

Note: these are general examples that may or may not be specifically used in research. 

Example 1: Disease Modeling

In its most fundamental use, CRISPR can damage specific locations in the genome (The genome is made up of all the genes and DNA in an organism).  When the CRISPR materials are introduced to the cells, they will come together to form a unit, move to the target location in the genome based on the recognition sequence, and the Cas protein will cleave the DNA – think like scissors cutting the DNA.

Now, you can imagine that having a cell’s DNA cleaved (cut) would not be very good for it, and you’d be right to assume that.

Fortunately, our cells have repair functions to be able to put two ends of DNA back together to restore function.  However, it doesn’t do this perfectly and sometimes there will be small insertions or deletions of DNA bases at the cut site.  These small insertions or deletions are called indels and will likely reduce or eliminate the functionality of the gene: this means that the gene won’t work properly.  This type of experiment is called a knockout because it “knocks out” a gene.

How does this work in a scientific experiment? In our example, we knock out SLC13A5 to model the disease in our dish of cells. Scientists can study how the absence of SLC13A5 affects different types of cells, like those in the brain, liver or bone. This is important because sometimes a gene works slightly differently in each cell type. Cells without a functioning SLC13A5 gene are unable to transport citrate across the cell membrane, and testing knockouts can answer questions like:

• Is cell growth affected?
• Do cells still proliferate?
• Do cells change their shape?
• Does the lack of this gene have an effect on other genes?
• Do mutations in different locations in the SLC13A5 gene have different effects?

Example 2: Targeted Gene Editing

Knocking out genes with indels is useful but limited: it may not edit all of the intended cells (low efficiency), or the guide RNA may velcro onto a different gene (low specificity). So scientists have modified the “scissors” of CRISPR to improve these factors in cell and animal models.  Two recent examples are called Base Editors and Prime Editors. These use a guide RNA to locate a specific sequence in the DNA, but instead of cutting the DNA to knock out the gene, they can make specific changes to the DNA.

In many SLC13A5 patient variants, a single DNA base is substituted for another, so knocking out the gene might not model the disease.  If we want to study the specific mutation a patient has, we need to be able to change a specific base. In this case, we can introduce these new CRISPR ingredients along with a guide RNA targeting our desired gene and make the change so that our plate of cells have exactly the same DNA sequence as a specific patient.

If we do this with multiple patient variants, it can shed light on the connection between gene behavior and patient symptoms.  This is important because we know of over 50 different SLC13A5 variants (changes to the SLC13A5 gene) that lead to SLC13A5 Epilepsy.

Gene Therapy

There are several types of gene therapies.  One type is CRISPR gene editing therapy.  Other types include CAR-T and AAV gene therapies.  To learn more about these different therapies, you can read this Science Simplified post from June 2021. Although TESS is not currently pursuing a CRISPR gene therapy for SLC13A5 Epilepsy, we are excited that these technologies to revert a mutated gene back into a healthy state are on the horizon.  CRISPR gene therapy may present many challenges. Even a high efficiency, highly specific gene therapy still needs to be administered in a way that it accesses the right cells, and regulations around gene therapy trials are still being established.  It’s also important to note that targeting each of the over 50 known mutations in SLC13A5 Epilepsy may not be the most effective approach: This is why TESS’ shots on goals include drug repurposing in addition to gene therapies. But researchers and clinicians around the world are tackling these problems, and we have more promising advances than ever before.

I hope this brief overview of CRISPR highlights its utility in research and medicine; it is one of our best tools for gene editing and serves as a foundation for future techniques.  It allows for manipulation of the genetic code with more precision than ever before and can easily target many different genes and even different regions of the same gene.  However, it is also important to remember that there are still many limitations to its use for therapeutics, which is a topic that will be expanded upon in a future post.