Genome editing and stem cells: Questions and Answers

Genome editing is the process through which a piece of DNA in any cell (plant, animal, yeast, bacterial) is removed, replaced or added. If for a moment we think of the genome as a large book, ‘editing’ would involve changing single letters in that book such that a new word is produced. That is the level of detail required in genome editing. The editing process is based on “scissor” proteins that can recognize a specific piece of DNA amongst the entire genome and make a break in the double helix. The cell machinery usually repairs this break but occasionally there is an error in the repair that changes the DNA sequence in the cell. Scientists use this process in genome editing by engineering the “scissor” proteins to make precise cuts that will result in genes being switched off. Alternatively, they can add in DNA code. For example, they can prompt cells to incorporate new code at the “break spots” to substitute a mutated gene for a healthy one or vice versa.

Up to very recently, researchers edited DNA with techniques that were difficult and time consuming requiring years even for a very small genomic edit. However, a new genome editing tool, called CRISPR/Cas9, discovered by studying the world of bacteria, is making editing fast, simple and precise and therefore the real potential of genome editing is now at hand.

Genome editing has been around in a number of forms for decades. The first generation was based on molecular “scissors”, such as ZNFs and TALENS, that could make cuts at specific locations, but their design was complex, slow and not very specific. However, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a new technology for genome editing that is simply designed, fast, and precise. It is based on an ingenious natural mechanism that bacteria have developed to fight against viruses. Scientists learned the process from the bacteria and adapted it to create a technique that can be used in the lab to engineer the genome of many species, including humans.

In a nutshell, the tool works like this: Cas9 is like a pair of molecular scissors that is positioned by a guide RNA (a molecule very similar to DNA) at a precise location that is complementary to the code of DNA the scientists wish to target. The Cas9 “scissors” then cut the DNA. After the cut is made, the cell recognizes it as a natural mistake and begins to repair the DNA. More often than not, the cell will repair the cut. However, there will be instances in which the cell does not repair the cut correctly which, in turn, turns off the targeted gene’s function – the desired result. Alternatively, this technology can be used to insert new piece of DNA to replace a gene. Many sophisticated changes can be done with this selective targeting.

In many ways, genome editing represents a scientific revolution for the entire field of biological sciences – from synthetic biology to plant science to stem cell research. Genome editing has been done for decades with other techniques, but the process was laborious, slow, inefficient, and expensive. With the new generation of genome editing based on CRISPR/Cas9, scientists now have the tools to study the genome of humans and other species in detail and open unprecedented possibilities for therapy.

The most impactful change with the CRISPR/Cas9 process is the ability to delete or replace genes precisely and permanently with a simplicity that was not possible before. In this way, Scientists can investigate the function of particular genes and design new therapies, including gene therapy based on gene correction. For example, genome editing can help engineer cells of the immune system recognize and destroy cancer cells.

Additionally, much attention has been given to the application of genomic editing in stem cells as well as the concerns and possibilities of editing human embryos for research or clinical use.

A genetic modification is any manipulation that produces a change in the genome of a living organism. It is often called genetic engineering. A genetic modification works by adding a set of genes generated outside the organism (of the same species or different species) to the genome in order to obtain a specific effect for research, medical, agricultural, and other scientific goals.

Genome editing is a type of genetic modification based on genome targeting that uses molecular “scissors”, such as CRISPR/Cas9. The main difference with genome editing is that the hosts own DNA is selectively altered at specific locations. In other words, with genome editing we don’t just add new sentences to a book, but swap around the book’s own words with precision.

The majority of the cells that form your body – from blood to skin – are called “somatic cells”. They include tissue (‘adult’) stem cells that help regenerate tissues. The genetic information of somatic cells will not be passed down to a new generation. Therefore, a somatic cell gene modification – like the engineering of bone marrow cells – will not be inherited by future generations.

Germ-line cells including eggs, sperm and of cells of the embryo do transmit their DNA from generation to generation. In fact, every cell of their resulting offspring will inherit the cell’s genetic material. Therefore, germ-line genome editing will affect the whole individual’s genome and will be permanent through new generations. For example, repairs to a familial (‘heritable’) genetic disease in a fertilized egg will be carried forward in the DNA of all subsequent cells of the offspring formed from that fertilized egg.

Theoretically, any type of cell with DNA can be edited. So any type of stem cell can have its genome edited. This includes:

• tissue (adult) stem cells;
• germline stem cells (sperm cells, oocytes/eggs) and cells in the early embryo;
• pluripotent stem cells (such as embryonic stem cells and induced pluripotent stem cells).

Whilst stem cells within the early embryo can potentially be edited for research studies, these studies are done under rigid ethics and regulations. At the present, it’s illegal in the UK and many countries to edit both human germ line cells (sperm and oocytes/eggs) and embryos for clinical use and reproduction.  In summary, if the purpose of the editing is research only, the procedure is permitted with the appropriate regulatory approval. However, if the purpose of the editing is clinical use or reproduction, the procedure is illegal.

Being able to genetically change stem cells offers an extraordinary tool to advance both basic research and therapy. Scientists want to study how stem cells and their genome work. To achieve this, they need to make changes in the genes to discover their role and the ways in which they may be involved in human development or the development of disease.

Genetically modifying stem cells also opens up new opportunities for researchers to model diseases in the lab. Since stem cells can be turned into desired cell types, genetic modification allows re-creation of the “disease in the petri dish”.

Clinical use of genetically edited stem cells derived from patients is another area of research with strong potential. Tissue (‘adult’) cells can be taken from a patient affected by a genetic disorder and reprogrammed into ‘induced pluripotent stem cells’ (iPS cells). iPS cells (like embryonic stem cells) have the ability to form all the cell types in the body. These iPS cells can be edited with CRISPR to repair the genes causing the disease in the patient. Finally, these disease-corrected pluripotent stem cells are specialized (‘differentiated’) into the desired cells that are required for transplantation. For example, scientists were able to restore the muscle function of mice affected by fatal Duchenne muscular dystrophy. To do this, they extracted stem cells from patients with Duchenne’s, genetically correct these pluripotent cells, and created healthy muscle cells. These healthy cells were then introduced to the affected mice resulting in the restoration of muscle function.

Advancing blue sky research and development biology

With the advancement of research with human pluripotent stem cells, scientists are recreating some key steps of human development in the laboratory. However, there is still much to be understood.

Genome editing enables investigation of biology and human development in many new ways. By switching developmentally important genes on and off, researchers are able to track their function within the greater organism. It also allows researchers to target multiple genes at the same time to understand how they work together as a complex network. Greater understanding of the underlying cell biology and development processes could help in the discovery of new therapies for a wide range of diseases as well as the development of better protocols for cellular replacement therapies based on stem cells.

Studying the make-up of DNA epigenetics

Genome editing could change the branch of research that studies the chemical modifications that sit on top of DNA, like flags, that modify the activity of genes. These changes are called ‘epigenetic modifications’. Many diseases including cancer and syndromes caused by chromosomal abnormalities are associated with epigenetic changes. For the first time scientists are now able to engineer these chemical modifications on the DNA using CRISPR/Cas technology. They are then able to test the function of these flags in complex diseases and stem cells.

Revolutionizing drug discovery

Part of the challenge in the discovery of new drugs is the identification of new therapeutic targets. A common strategy involves screening large numbers of mutated genes in the search for potential therapeutic targets. In this way, scientists can pin-point, amongst thousands of genes, those responsible for a disease related effect. This leads them to new targets for drug treatments and, ultimately, the development of new medicines.  To increase their chances to find new targets, scientists must have the ability to investigate the function of genes at a large scale – ideally including the ~24,000 genes that comprise the entire human genome

Genome editing revolutionizes this quest for scale in a number of ways. The ease of the process allows for the editing of thousands of genes in parallel at speeds that would have been impossible with the tools available before CRISPR. Therefore, genome editing holds the potential to reduce the time it takes to develop new drugs as well as the costs associated with the process. Scientists are already taking advantage of this potential. For example, the technique has been used to screen the entire human genome for the genes that give cancer cells resistance to a particular chemotherapy drug. This research opens up new options for the selection of treatments for people affected by cancer as well as new avenues for developing therapies.

Powering disease modelling

Genome editing is made a more powerful research tool when combined with disease modeling. Advances in stem cell research has allowed us to understand the progression of human diseases in a dish by making human iPSC cells specific to a condition and then, by inducing specialization (‘differentiation’) into the desired cells we want to study, ultimately modeling the disease progression. However, the comparison of healthy models and disease models from donors with different genetic backgrounds makes robust data interpretation tricky.

With genome editing, scientists can specifically repair a disease-causing mutation in a patient’s cells and, importantly, compare their biology to an unchanged control. This allows scientists to generate healthy and disease models of complex multi-genetic diseases with the same genetic background. Likewise, the process can be adopted also in the opposite way by inserting mutated genes into healthy stem cells to see if they develop the disease.

These studies have the potential to increase understanding of how genes affect disease progression and provide an ideal system for testing drugs and therapies. Currently, several groups are now using CRISPR to model complex neurodegenerative diseases such as Alzheimer’s.

The basic idea of genome editing is to repair a disease-causing gene directly where it is mutated in the DNA of the patient’s cells. Therapeutic gene targeting approaches have already been tried with earlier gene editing techniques and some are in clinical trials. However, new genome editing tools (like CRISPR) allow for more accurate and faster editing and permits the editing of many genes at the same time which is important for complex multi-genetic diseases.  

Researchers are producing proof-of-concept studies for genome editing use in gene therapy both ‘in vivo’ (editing of genes within the body) and ‘ex vivo’ (editing of genes in the laboratory before re-introduction of the cells to the body). The ‘ex vivo’ approach can be done in two ways. In the first, the patient’s specialized cells are collected, corrected in the lab, and re-injected into the patient. In the second way, somatic (‘adult’) cells are collected from the patient. In the lab, these somatic cells are then “reprogrammed” into induced pluripotent stem cells and the specific gene is corrected. The induced pluripotent stem cells are then specialized into the cells of interest and, finally, re-injected into the patient.

There are several positive examples of research using this therapeutic approach:

• There is currently no cure for HIV infection; drug-based treatments can only halt the progression of the disease. Scientists have tested a new route for removing latent HIV entirely by removing the virus DNA from the genome of immune cells that have been corrected by CRISPR genome editing.
• Epithelial stem cells taken from people with cystic fibrosis have been used to make small organs (‘organoids’) that model the disease in the laboratory. Somatic (‘adult’) cell gene correction using CRISPR technology was used to restore physiological functions lost in cystic fibrosis. Further information on stem cells organoids and genome editing can be found in this review.
• A group of scientists have corrected a blood genetic disorder, B-thalassemia, in patient-derived induced pluripotent stem cells. They were then able to generate red blood stem cells from these corrected stem cells. This therapy  could be used for cellular replacement in anemic patients.  

Clinical trials considering the risks and benefits of these approaches will clearly need to be performed before the new generation of therapeutic genome editing reaches the clinic.

There are very good reasons to think that genome editing could help patients in the near future. Although it is not currently being used in treatment for any disease, genome editing allows scientists to conduct better research across the spectrum of genetic diseases.

Many groups around the world are working with this powerful technology and the scientific community expects that genome editing will help discover new treatments for genetic diseases in the not-so-distant future. This will be achieved in different ways from the discovery of the molecular mechanisms that control a disease via large drug screening to disease modeling and gene therapy. Researchers are also trying to improve the reliability and safety of genome editing in order to apply this futuristic technology to treating genetic diseases directly.   

The number of researchers working with human embryos is very limited compared to those studying stem cells or somatic (adult) cells. However, for many scientists, there are a multitude of research areas that could lead to treatments with the use of genome editing in embryos. These include: prevention of inheritable genetic diseases in offspring of at-risk parents, correction of infertility genes in the sperm or oocytes of parents, research for advancement in assisted reproduction technologies and pre-implantation genetic diagnosis.

Some scientists want to edit the genome of embryos in order to understand the biology of early human development and help improve assisted reproduction technologies. In this case, any genome editing would have to be performed in embryos that will never be implanted and result in pregnancy, in order to comply with current regulations.

Other scientists want to explore the feasibility of the technique for gene correction in embryos of high-risk parents carrying an inheritable genetic disease (such as Huntington’s Disease). Genome editing of embryos in this case would prevent the disease mutation from being passed down to the at-risk parent’s future children. 

The prospect of manipulating the genome of human embryos has raised debates and discussions amongst scientists, regulators, and the public. It is a crucial conversation that we need to participate in as we have done for other scientific advancements in the past from embryonic stem cells research to ‘in vitro’ fertilization and, recently in the UK, mitochondrial replacement.

So, what are the ethical concerns? It depends on an important distinction, that is, if we are talking about a genetic manipulation that has a research, clinical or reproductive goal. This distinction is explored in our dedicated article “The ethics of changing genes in the embryo”.

For research purposes, scientists need to have permission from an ethics committee in order to obtain and modify the human embryo. Crucially, no baby will be born as a result of this research.

For reproduction and clinical purposes, genome editing of embryos is banned (either through law as in the UK, Europe, Canada, Australia or through guidelines as in China). The issues cited in these regulations include the safety of this manipulation for future generations, the potential risk for the health of the person and the population as a whole. Some people also point to the argument that genome editing for non-research purposes could be a slippery slope towards ‘designer babies’ with enhanced characteristics of non-medical traits. However, to put the latter point into prospective, even if genome editing for desired traits were legal, the science is far beyond what is currently available. Even simple physical characteristics, such as hair colour, are extremely complex and manipulation of these simple characteristics are currently beyond the understanding of the scientific knowledge. Traits, like intelligence, that are mainly the result of a combination of genetics and nurture are far more complex than simple physical traits and therefore farther beyond the realm of feasible manipulation.

There are a number of important ethical issues, both scientific and moral, with germ line modification. These concerns are taken very seriously by scientists around the world. In December 2015, a group of scientific organizations from the UK, China and the US convened in Washington for a global summit on human gene editing and have published ethical recommendations. The main concerns about germ line modification stated in the summit report were: 

• the risks of inaccurate editing and incomplete editing of the cells of early-stage embryos;
• the difficulty of predicting harmful effects that genetic changes may have under the wide range of circumstances experienced by the human population;
• the obligation to consider implications for both the individual and future generations who will carry the genetic alterations;
• the fact that, once introduced into the human population, genetic alterations would be difficult to remove and would not remain within any single community or country;
• the possibility that permanent genetic ‘enhancements’ to subsets of the population could exacerbate social inequities or be used coercively; and,
• the moral and ethical considerations in purposefully altering human evolution using this technology.

They conclude that “it would be irresponsible to proceed with any clinical use of germ line editing unless and until (i) the relevant safety and efficacy issues have been resolved, based on appropriate understanding and balancing of risks, potential benefits, and alternatives, and (ii) there is broad societal consensus about the appropriateness of the proposed application.”

This meeting was followed in January 2016 by a stem cell-focused workshop on genome editing organized by The University of Cambridge, which has recently published a summary report.

A very important message that came from the meeting is the recognition that policy development in this area should be the product of a concerted and consultative strategy that actively involves the public as well as policy makers and scientists.

For clinical use, genetic modification of the human germ line is officially banned in the UK, Europe, Canada, Australia, China (via guidelines) and in the US it is restricted but a comparable legislation is not in place. Many other countries are following with similar laws. Clearly, a comprehensive and international regulatory architecture, promoted by international scientific and health organizations, such as the World Health Organization (WHO), will need to be built to keep up with the scientific advancements in this area.

For research purposes, germ line genome editing (in early embryos that will not be used for reproduction) is moving ahead under strict regulations. Recently, the UK Human Fertilization and Embryology Authority (HFEA) approved a group of scientists at the Francis Crick Institute, London, to use CRISPR to conduct research in early embryos that will be stopped at 250 cells. Scientists on the project, hope that this work – the first of its kind – will improve the success rate of in vitro fertilization through improved understanding of the biology of early human development.

The UK has an advanced juridical system on embryonic research due to its pioneering role in this field and has adopted a case-specific approach to this delicate subject. For example, recently the UK approved the clinical use of mitochondrial DNA replacement therapy. This consists of the transfer of the nuclear genome from an egg containing unhealthy mitochondria into a donor healthy egg, thus leaving the unhealthy mitochondria behind. This technique will be allowed on a case-by-case basis to prevent the transmission of life-threatening mitochondrial diseases from parents to children. More information can be found on the report published by the HFEA, which also includes public consultation findings.

In other parts of the world, regulation of germ line research involving genome editing is more nuanced. A Chinese group reported the gene correction of a blood-associated disease in human embryos that were ‘non-viable’ (embryos that could not lead to a birth if implanted). Since the study had in mind a therapeutic goal and not reproduction, it remains in the remit of research. This means that the study does not breach the established germ line ethical guidelines, even if it sparked much debate. This study is controversial not because of the findings of the study but because it suggests a clinical route for the application of germ line genome editing that many in the scientific community feel is premature.

No. The vast majority of embryonic stem cell research does not require the manipulation of embryos. Many groups work with embryonic stem cells derived from embryos that could not be implanted after ‘in vitro’ fertilization. Laboratories that concentrate on the study of embryos are very closely linked to ‘in vitro’ fertilization units and their work follows strict regulations, oversight and ethical guidelines. For more information, see our factsheet on the origins of human embryonic stem cells.