What we talk about when we talk about CRISPR

Written by EpiPredict PhD fellow, Emre Sofyali (DKFZ, DE), together with EpiPredict Partner Organisation Science Matters. The EpiPredict consortium receives funding from the EU Horizon 2020 program under Marie Sklodowska-Curie grant agreement no. 642691.

It has not been very long that the world got introduced with the concept of CRISPR — a revolutionary approach on editing the genome. CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats. Mouthful, right? Well, basically CRISPR derives its name from the unique stretches of DNA (our unique genetic material) that it targets. These short stretches of DNA are recognized by a special molecule (guide RNA) that forms a complex with an enzyme (Cas9) to bind and induce a cut at the recognized DNA region. The CRISPR system is derived from bacteria where it recognizes virus DNA and digest it into small pieces to make the virus harmless. Now, we use CRISPR in our advantage and specifically engineer the guide RNA to recognize a desired DNA location and make a cut there. This cleavage results in a break in DNA which can be repaired by the cell. However, this break in the DNA helix enables the researchers either to delete or insert pieces of DNA code from or into the target region, respectively (genome editing).

We can think of CRISPR-Cas9 system as microscopic scissors with an incredible precision. Precision is the key word for this approach since editing of the genome with this level of specificity was not possible before CRISPR. And precision is achieved by aforementioned guide RNA (gRNA) and Cas9 complex that scans through the entire genome to find the exact matching sequence between gRNA and target DNA. We can say that gRNA acts like a GPS to find the ultimate destination through the intricate highways of the genetic material. When the complex reaches the destination, the Cas9 enzyme cleaves the double helix of the DNA and the edits are introduced in the genome.

Image by Science Matters

This is not the only approach for genetic engineering that CRISPR system has to offer. There are many studies where the so-called GPS feature of enzyme-gRNA complex is used to recruit proteins. However in that case the cutting properties of Cas9 are disabled (dCas9 — stands for deactivated Cas9). Instead the dCas9 is coupled to proteins that have the potential to activate or inactivate parts of the DNA. Before giving an example for this aspect of CRISPR approach let me tell you a little bit more about our research.

In the frame of the EpiPredict consortium, we make use of the CRISPR system by utilizing it as a tool for interfering with the layer on top of genetic code. We will refer to this layer as epigenetic layer from now on just to keep the fancy mouthful scientific jargon intact. Well, if we try to explain simply, genes in our DNA code for the proteins which in turn perform functions to sustain life. There are many factors that may affect the activity of a gene and one of these factors is the environment in which a gene is located. Different environments in the DNA may sound a bit strange, but you have to know that the DNA strand is not just a long stretched out strand, but it is wrapped around proteins which can be chemically altered to allow or block access of certain DNA regions. Also the DNA itself can get chemical modifications that may affect its activity. If you wish to know more about what epigenetics is in a nutshell, here is a very nice blog that might be considered as “Introduction to Epigenetics” written by fellows in our project consortium.

In our project, we are mainly interested in uncovering what underlies the resistance to hormone therapy in a certain subtype of breast cancer. In this case the therapy is working at first, but after a while the treatment response is lost. We have reasons to believe that alterations in the above mentioned epigenetic layer might play an important role in this development of resistance against the hormone treatment. That is why we are looking for therapeutic cues to interfere with this layer aiming to reverse the resistance and re-sensitize the cancer cells to the therapy. For that, we perform epigenetic interference by using the highly specific CRISPR/dCas9 tool. This system enables us to temper with the modifications on or around the DNA and thereby change activity of genes in a targeted manner.

Still confusing, right? Let me try to elucidate with an example:

Imagine gene X is known to be impaired in the acquired resistance against the hormone therapy and re-activation of gene X is able to return sensitivivty to the therapy which means a patient can benefit again from the conventional therapy. To induce the activity of gene X we can target the region of DNA that codes for gene X with CRISPR/dCas9. In this way dCas9 fused with specific epigenetic marker modifiers (epi-modifiers) is recruited. The attached epi-modifier removes modifications associated with repression and/or introduces modifications associated with activation. Hereby we change the genomic environment of gene X resulting in re-activation.

Image by Science Matters

Targeted re-writing of epigenetic modifications might give the opportunity to alter or fine-tune the activity of target genes. Although there are many technical hurdles that need to be overcome about the technology itself and ethical questions regarding usage of CRISPR in treating human diseases, it is beyond any question that this cutting-edge technology has a lot to offer in terms of (epi)genetic editing.

Epigenetic based prediction of therapy resistance in breast cancer: an EU Marie Skłodowska Curie Innovative Training Network