In 2012, the teams of Emmanuelle Charpentier (University of Umea, Sweden) and Jennifer Doudna (University of California, Berkeley, USA) described a new tool to edit the genome. This innovation, called CRISPR-Cas9, had an immediate impact and rapidly established itself in research laboratories. Today, the Institut Curie is opening a platform dedicated to the application of this technology to genetic screening.
The CRISPR-Cas9 technology
CRISPR-Cas9 works like a pair of scissors capable of cutting the genome precisely. This technology is based on a complex composed of a small RNA called “guide” and the nuclease Cas9. The complex thus formed binds to a specific DNA sequence, complementary to the guide RNA. This binding is followed by a double strand cut of the DNA by Cas9. DNA repair mechanisms can subsequently be used to introduce precise mutations. In this way, researchers can manipulate DNA to suppress the function of a gene or replace it with a modified gene (the so-called “homologous recombination” method).
CRISPR-Cas9, a new technology? The CRISPR system (Clustered Regularly Interspaced Short Palindromic Repeats) has been around for millions of years. Bacteria use it as an adaptive immune defense mechanism against viruses. The methodology is simple and extremely effective: upon entry into bacteria, the viral genome is recognized by this dedicated immune memory system. Bacteria then destroy the genome of their enemy by cutting it off.
The use of programmable nucleases for genome-modifying purposes has been underway for about 12 years in laboratories. However, first generation nucleases were very complex to implement. CRISPR-Cas9 is revolutionary in its ease of programming, speed of execution and lower cost compared to other nucleases. The high flexibility of the CRISPR-Cas9 system makes it possible to perform genetic screening experiments. This methodology entails mutating all known genes in a population of cells, each cell carrying a single mutation. It is then possible to determine which genes are involved in a biological process of interest (cell growth, resistance to treatments, etc.).
A new genetic screening platform based on CRISPR-Cas9 has just been created at the Institut Curie’s research center. The CRISPR’it platform has generated a lot of enthusiasm.” We are already working with 15 research teams encompassing various topics “, says Camille Fouassier, the platform manager.
The evolution of the technology
The CRISPR-Cas9 technology has still important limitations. It can only be used at certain sites in the DNA sequence. And the sgRNA/Cas9 complex can cut sequences that resemble the target sequence (so called ‘off targets”). In addition, DNA cutting can induce a state of cellular stress and therefore bias the analysis of the resulting phenotypes. Finally, the DNA repair process leads to highly variable type of mutations that do not always eliminate gene function.
To overcome these limitations, scientists are working on derivatives of the CRISPR-Cas9 system. Alternative approaches being studied include a more precise and less toxic spinoff that introduces DNA substitutions. In particular, this method has the ability to change a codon into a premature stop signal for protein synthesis (“stop” codon).
Other derivatives take advantage of the opportunity offered by the CRISPR-Cas9 system to target a DNA sequence of interest. Instead of using CAS9 nuclease activity to edit the DNA sequence, local CAS9 recruitment can be used to modulate the overlying biological processes. For example, it is possible to increase or decrease gene expression or to locally modify the chemical composition of DNA or of the histone proteins associated with it. These chemical changes in turn influence different biological processes such as DNA repair or gene expression.
Applications of CRISPR-Cas9 at the Institut Curie
The applications in research are immense, since biologists now have the ability to introduce all types of genetic modifications into cells or model organisms. In just a few years, CRISPR-Cas9 has become a central tool in many research programs.
In particular, genetic screening by CRISPR-Cas9 represents a key asset for cancer research. For example, it is possible to address the problem of resistance to anti-tumor treatments. Genetic screening can be used to identify genes whose mutation makes cells resistant to a therapeutic molecule. The technology is also used to identify the Achilles heel of each type of cancer using a synthetic lethality approach. This latter strategy is based on the assumption that mutations that cause cancer create new vulnerabilities that can be used to kill cancer cells. Although CRISPR-Cas9 screening is most often deployed in in vitro cell culture systems, the ambition of the new platform is to work as closely as possible to the natural tumor environment. In particular, 3D culture systems (known as organoids), which recreate in vitro part of the architectural properties of tumors, represent attractive model systems. An approach also under investigation is the use of genetic screens on patient-derived xenografts, which currently represent the best models of cancer for pre-clinical studies.
Another application of the technology is the study of recurrent mutations found in cancer. These mutations can lead to an increase in the activity of oncogenic proteins or, conversely, a loss of the function of proteins that inhibit tumor development. However, we still poorly understand the function of most mutated genes in cancer.” By reintroducing these genetic abnormalities into cell lines thanks to CRISPR-Cas9, researchers are able to evaluate their impact on tumor development”, said Michel Wassef, scientific co-director of the platform.
Applications for immunotherapy are also under development. T lymphocytes taken from patients are genetically modified to express a chimeric receptor programmed to recognize a tumor marker (CAR T cells) and thus destroy the diseased cells.