CRISPR/Cas9 – Rewriting the code of life

The discovery of CRISPR/Cas9 genetic scissors has revolutionized a lot of research areas, not least within life sciences, and the technology is bringing hope for new cancer therapies and the treatment of inherited diseases.

The 2020 Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer Doudna’s discovery of CRISPR/Cas9 genetic scissors. This enzyme system, which utilizes a very delicate and targeted mechanism to cleave DNA and insert new DNA parts, holds enormous power that affects us all, stated the Nobel Committee for Chemistry announcement.

“Within basic research CRISPR has revolutionized the simplicity, for example in identifying genes that affect different biological processes. Within clinical research we are starting to see the first examples of how rare monogenic diseases probably could be cured by using CRISPR to affect the disease-creating mutations. This fundamentally changes how we envision the future of healthcare.”

The groundbreaking factor in this discovery is in short that it is so easy to alter genes in a cell. Previously this would take a very long time, it was expensive and precision was low, explains researcher Fredrik Wermeling, PhD, at the Department of Medicine at Karolinska Institutet in Solna.

“Within basic research CRISPR has revolutionized the simplicity, for example in identifying genes that affect different biological processes. Within clinical research we are starting to see the first examples of how rare monogenic diseases probably could be cured by using CRISPR to affect the disease-creating mutations. This fundamentally changes how we envision the future of healthcare.”

Left: Illustration of CRISPR/Cas9. Right: Fredrik Wermeling, Assistant professor, Karolinska Institutet. Photo: Erik Holmgren

Reshaping life sciences

Ever since the molecular structure of DNA was reported in 1953, scientists have been trying to manipulate genes in cells and organisms. Down the years, important findings and advancements have been made, eventually leading to this year’s Nobel Prize and opening the door to this enormous potential to rewrite the code of life.

These findings include the discovery of unusual repeated structures common in procaryotes’ genomes containing the same features, suggesting an ancestral origin and high biological relevance, and the introduction of the term for these, CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats. Both the repeats and the spacer sequences between them, remnants of genetic code from past invaders, DNA remnants or DNA scars, gave the scientists more clues and they came to the conclusion that CRISPR was in fact the bacteria’s immune system against virus, and that bacteria had memory. It was discovered that bacteria transcribe these DNA elements into RNA upon viral infection. The RNA guides a nuclease (a protein that cleaves DNA) to the viral DNA to cut it, providing protection against the virus. The nucleases are named “Cas”, meaning “CRISPR-associated”.

”This is an excellent example of how basic science research on RNA in a humble bacterium could facilitate crucial development of biotechnologies, and ultimately treatment of diseases. The technology is now used in almost every scientific field, ranging from basic to translational research.”

Emmanuelle Charpentier discovered a previously unknown molecule tracrRNA when she studied streptococcus pyogenes, and she showed in 2011 that this molecule is part of the CRISPR/Cas system. Emmanuelle Charpentier and Jennifer Doudna managed to decode the functions of the repeated DNA sequences (CRISPR) together with Cas (CRISPR-associated) proteins. They were able to recreate the bacteria’s genetic scissors in a test tube and the two scientists simplified the molecular components of the scissors so they were easier to use. They had uncovered a fundamental mechanism in a bacterium that causes great suffering for humanity. But it did not stop there, they were also able to reprogram the genetic scissors so that they could cut any DNA molecule at a predetermined site. They demonstrated that RNAs could be constructed to guide a Cas nuclease (Cas9 was the first used) to any DNA sequence. In their game-changing paper they concluded that there was “considerable potential for gene-targeting and genome-editing applications”. Now, just eight years later, their discovery has literally reshaped life science.

”This is an excellent example of how basic science research on RNA in a humble bacterium could facilitate crucial development of biotechnologies, and ultimately treatment of diseases. The technology is now used in almost every scientific field, ranging from basic to translational research,” says Edmund Loh, researcher at the Department of Microbiology, Tumor and Cell biology at Karolinska Institutet, and a collaborator and friend of Emmanuelle Charpentier.

Left: Edmund Loh, Principal investigator, Karolinska Institutet. Photo: Francesco Righetti. Right: Illustration CRISPR/Cas9

There are now a number of different CRISPR/Cas systems known and these are divided into two major classes. In the Class 1 systems, specialized Cas proteins assemble into a large CRISPR-associated complex for antiviral defense (Cascade). The Class 2 systems are simpler and contain a single multidomain crRNA-binding protein (e.g., Cas9) that contains all the activities necessary for interference, described the Nobel Committee for Chemistry. The system has been found in around 40 percent of all known bacteria and even 90 percent of all known archaea. Each system has a different protospacer adjacent motif (PAM). This motif is the only absolute requirement for CRISPR to work.

“Hopefully, the discovery will generate more attention from policy makers such as the government, pharmaceutical industries and philanthropic foundations to focus on and fund basic science research.”

In short it works like this. When a researcher aims to edit a genome they artificially construct what is known as a guideRNA (gRNA), which matches the DNA code where the cut is to be made. The scissor protein, Cas9, forms a complex with the gRNA, which takes the scissors to the place in the genome where the cut is to be made.

“I think the CRISPR/Cas9 discovery and technology benefit all life science fields. In addition, the background story and discovery could encourage more young people and women to be interested in basic science. Hopefully, the discovery will generate more attention from policy makers such as the government, pharmaceutical industries and philanthropic foundations to focus on and fund basic science research,” says Edmund Loh.

Exciting possibilities and realities

Since the discovery the field has exploded with applications and CRISPR has become a cost-effective and convenient tool for many different purposes. It can be used for genome editing (knockouts, knockins, exchange of base pairs, removal of genetic elements, homologous recombination) and gene regulation using CRISPR activation (attraction of transcription factors) and CRISPR inhibition (usage of KRAB repressor). It can also be used for tagging genetic elements, reporters, and for functional studies it is possible to have inducible CRISPR systems. According to the Biomedical Centre at the University of Iceland, it may also be used for dynamic imaging of genomic loci in living cells (comparable to FISH, without the need of cell fixation), and can be used in all cells of all organisms.

The development potential of the CRISPR system is also enormous and scientists all over the world are making progress almost every day. Just last year, a person with a genetic condition that causes blindness became the first person to receive a CRISPR/Cas9 gene therapy administered directly into their body. The treatment is part of a landmark clinical trial to test the ability of CRISPR/Cas9 gene-editing techniques to remove mutations that cause a rare condition called Leber’s congenital amaurosis 10 (LCA10).

“A third example is gene therapy for different severe inherited monogenic diseases related to the hematopoietic system, such as sickle cell anemia, beta-thalassemia, SCID and WASP.”

According to Wermeling, another very relevant example of recent progress is the SHERLOCK method. “In this method a modified CRISPR system is used to quickly identify if a sample contains a specific nucleotide sequence. This method is used for example to identify if a sample contains SARS-CoV-2 and it can be used as a quick and sensitive diagnostic test.”

The possibility to tailor make organs for transplantation is another application that has huge potential to solve many of the challenges related to transplant care. Wermeling says, “A third example is gene therapy for different severe inherited monogenic diseases related to the hematopoietic system, such as sickle cell anemia, beta-thalassemia, SCID and WASP.”

CRISPR (= Clustered Regularly Interspaced Short Palindromic Repeats) + DNA fragment, E.Coli Deposition authors: Mulepati, S., Bailey, S.; visualization author: User:Astrojan – http://www.rcsb.org/pdb/explore/explore.do?structureId=4qyz

Scientists have already started some clinical studies and the initial results look promising, according to Wermeling. “This could hopefully open up for treatments of inherited monogenic diseases affecting other organs, for example cystic fibrosis and Huntingtons disease. In the long run, the possibility to treat more common and more genetically complex diseases, such as cardiovascular diseases and cancer, but also dementia, allergies and autoimmune disease, is of course very appealing. It is however, not as clear how these diseases may be tackled using CRISPR,” says Wermeling.

He also mentions exciting possibilities related to immunotherapy treatments in cancer, where immune cells are instructed to attack the patient’s tumor and metastases. “Clinical studies are already ongoing where the immune cells are modified with CRISPR to become more aggressive and resistant.”

“We are using CRISPR to inactivate genes in different cells, and hence identify how these genes are affecting different disease processes. We are doing this using pre-clinical models and patient material to try to identify new targets for pharmaceuticals to treat these diseases.”

In his own research, Fredrik Wermeling and his research group at Karolinska Institutet are studying the immune defense in relation to autoimmune diseases and immunotherapy in cancer.

“We are using CRISPR to inactivate genes in different cells, and hence identify how these genes are affecting different disease processes. We are doing this using pre-clinical models and patient material to try to identify new targets for pharmaceuticals to treat these diseases,” he explains.

Wermeling sees great potential in how he and his colleagues are using CRISPR screening to understand how cancer cells develop resistance against different cancer treatments. When a drug is administrated to a cancer patient single cancer cells that succeed in avoiding the negative effects of the treatment will have survival advantages.

“Through a classic ‘survival of the fittest’ evolution process, over time the patient is therefore developing tumors that are made of more and more cancer cells that are resistant against the pharmaceutical and eventually the tumor is not responding at all to the drug,” he says. “This is still a long way away, but to be able to use CRISPR screening to identify in what ways specific cancer cells avoid an initial effective pharmaceutical, and develop resistance, creates possibilities for highly effective combination treatments. There are conceptual similarities with the cocktail of three antiviral drugs that together effectively inhibit the life cycle of HIV, but where the virus quickly develops resistance if the patient is treated with only one drug at a time.”

Regulations are required

As with every powerful technology, the genetic scissors require regulation to avoid unethical applications. Causing changes in a germ cell or embryo, so that the change is inherited by coming generations, is far more controversial than editing the ordinary cells of a human being suffering from a genetic disorder via gene therapy. In 2018, CRISPR was for example used by the Chinese biologist He Jiankui to modify twin embryos used for IVF, resulting in the birth of two girls allegedly with alleles that would confer protection from infection by HIV. He bypassed ethical regulations and chose to use germline editing for pre-emptive protection, in addition, he did not show any clear evidence that the procedure was safe. Jiankui’s actions were condemned by the biological community.

The Nobel Committee for Chemistry also states, “Experiments that involve humans and animals must always be reviewed and approved by ethical committees before they are carried out.”

Another controversial aspect is the possibility of improving or refining perfectly normal human conditions with the help of CRISPR/Cas9. Perhaps adjustments in the genome might eventually lead to more intelligent, more productive or more beautiful human beings.

For many years there have been laws and regulations that control the application of genetic engineering. The regulations include prohibitions on modifying the human genome in a way that allows the changes to be inherited. The Nobel Committee for Chemistry also states, “Experiments that involve humans and animals must always be reviewed and approved by ethical committees before they are carried out.”

In 2017, a report from an international committee convened by the U.S. National Academy of Sciences (NAS) and the National Academy of Medicine in Washington, D.C., concluded that human embryo editing could be ethically permissible one day – but only in rare circumstances and with safeguards in place. “Those situations could be limited to couples who both have a serious genetic disease and for whom embryo editing is “really the last reasonable option” if they want to have a healthy biological child,” says committee co-chair Alta Charo, a bioethicist at the University of Wisconsin in Madison.

CRISPR/Cas9. Illustration: Innovative Genomics Institute