Once again, the Nobel Prize recognizes the most revolutionary and innovative scientific discoveries that have the highest potential to transform humankind. Drs. Jennifer Doudna and Emmanuelle Charpentier, both biochemists, were awarded the Nobel Prize in Chemistry 2020 “for the development of a method for genome editing,” known as CRISPR.
This RNA-guided tool allows to change specific DNA sequences of cells in a very precise way, as to reverse deleterious genetic mutations responsible for human diseases. Once the selected section of a DNA is cut, it can be replaced with new genetic information correcting the mutation or providing new functions. This technique was originally observed as a natural way for bacteria to fight off viral infections, but as Doudna and Charpentier elucidated the mechanisms behind this adaptive bacterial immune response, these scientists realized that the CRISPR machinery could potentially be used as a molecular scissor to edit the genome of agricultural plants, and to cure human diseases.
In only eight years, the Doudna and Charpentier’s publication about how Cas9 works has accelerated the already exciting and very fast growing field of CRISPR research. Yan Zhang, Ph.D., Assistant Professor, Biological Chemistry, Medical School at the University of Michigan (U-M), explains that “the topic itself is very intriguing on the basic science side. Imagining that bacteria have an adaptive immune system used to be a ‘crazy idea!’ And that this bacterial RNA-based system would have applications impacting society and humankind used to be fiction. It is the excitement about the huge potential of CRISPR that attracts many scientists.”
While CRISPR technology could be of immense benefits to humankind, all along, Doudna has emphasized the profound ethical implications of using such tools and the importance of distinguishing between genome editing in somatic cells to cure diseases in an individual, and germ line cell modifications that are passed onto future generations. The ethical issues in the latter case are quite deep and should be carefully controlled (see the National Academy of Sciences publication: Heritable Human Genome Editing, 2020).
The therapeutic applications of CRISPR-Cas9 technology are still at an early stage, with only a limited number of ongoing clinical trials. So far, the tool has primarily been used to modify cells ex-vivo (outside the body). Once edited, these cells are returned to the patient to help fight diseases such as cancer or sickle cell disease. As procedures are being developed for efficient and safe systemic delivery of the CRISPR reagents to patients, many options will open up to treat over 10,000 human disorders caused by single gene mutations. Other possible applications of the CRISPR technology include detection of the presence of viruses such as SARS-CoV-2 virus that causes COVID-19 disease (see our publication, RNA Translated, “2020, the year of the RNA viruses” for more information on RNA viruses).
At U-M, many members of the Center for RNA Biomedicine are actively studying or using CRISPR technology and expanding its applications.
As scientists keep studying CRISPR, they realize that the Doudna and Charpentier’s discovery of S. pyogenes Cas9 system is only a small occurrence in the natural world. The Cas9 protein is the scissor that cuts the genome at precise sequences when guided by specific RNAs. For example, Dr. Zhang is leading a team that researches the genetic and biological role of N. meningitidis Cas9, a protein about 25% smaller than many other Cas9 proteins. NmeCas9 allows to target not only DNA, but also messenger RNAs that carry copies of the DNA information. This smaller protein can cut target sequences very precisely without affecting other genomic regions. Its small size facilitates the delivery of all the CRISPR-Cas components into cells, tissues and animals. “Delivery is a big challenge for CRISPR, as well as for many enzymes and therapeutic drugs,” adds Zhang.
In collaboration with Dr. Ailong Ke’s group from Cornell University who has complementary expertise in biochemistry and structural biology, the Zhang lab also studies the CRISPR-Cas3 system. This type of CRISPR comprises more than half of all known CRISPR-Cas systems. Cas3, a nuclease and helicase fusion enzyme, is recruited to the target sequence defined by guide RNA, and then travels along the DNA genome, breaking up longer sections of the genetic material as it goes. It is compared to a “DNA shredder with a motor.” The actions of Cas3 leads to a heterogenous population of engineered cells with large chromosomal deletions. This makes the Cas3 technology a powerful screening tool in discovering large genetic elements important for a particular disease.
CRISPR tools are also considered for precision cancer treatments to avoid collateral damage to normal tissues, which is the major cause of toxicity of current cancer therapeutics. Mats Ljungman, Professor in the Department of Radiation Oncology and Co-Director of the Center for RNA Biomedicine, and his team are researching how to harness the CRISPR machinery to precisely and selectively kill cancer cells. “The trick was to find a target that was cancer-specific where we could direct the CRISPR machinery to and induce lethal DNA double strand breaks. The target we settled on was chromosome rearrangement junctions,” Ljungman explains.
Chromosome rearrangements is a hallmark of cancer that occur early in carcinogenesis and are typically present in the hundreds in cancer cells. They drive tumor development by inducing oncogenes (amplifications) and causing loss of tumor suppressor genes (deletions). Despite having known about cancer-specific chromosome rearrangements for over hundred years, no therapy has to date been developed to target them for cancer treatment. Now, with technology developments in DNA sequencing and the discovery of CRISPR, it is for the first time possible to exploit this cancer hallmark.
A chromosome rearrangement event places two DNA sequences, which are normally far apart, next to each other in the cell. It is this cancer-specific juxtaposition of DNA sequences that the new precision CRISPR approach developed in the Ljungman lab is exploiting. By bringing two parts of an endonuclease to sequences on either side of the DNA junction, they will dimerize and form an active “killing machine” that cleaves the DNA. They have shown proof-of-concept that active endonucleases can be assembled specifically in cancer cells leading to the cleavage of DNA and cell death. The next big hurdle will be to show that this will happen in tumor cells in vivo. “We are encouraged about recent breakthrough developments in delivery systems for CRISPR and we are now eager to test our precision CRISPR approach in vivo using these new delivery systems,” says Ljungman.
As well-recognized by The Nobel Committee, CRISPR technology is on the verge of revolutionizing medicine, and, at U-M’s Center for RNA Biomedicine, we look forward to our scientists’ unique contributions to this fast-growing RNA-driven field of medicine. Bacteria have given humankind the precious gift of CRISPR, and it is now up to us to put it to good use.