Biotechnology and Gene Editing (CRISPR)

A Q&A with bioengineer Stanley Qi on the past, present, and future of CRISPR.

Over the past decade, CRISPR has revolutionised the biomedical world and the life sciences with its ability to easily and precisely edit DNA. Here, Stanford University bioengineer Stanley Qi explains how CRISPR works, why it’s such an important tool, and how it could be used in the future, including current developments in using CRISPR to edit the epigenome, which involves altering the chemistry of DNA instead of the DNA sequence itself.  “CRISPR is not merely a tool for research. It’s becoming a discipline, a driving force, and a promise that solves long-standing challenges from basic science, engineering, medicine, and the environment,” said Qi, an associate professor in the Department of Bioengineering and Institute Scholar at Sarafan ChEM-H. “Together, we can think innovatively about how to match needs with technologies to solve the most challenging problems.”

What is CRISPR?

CRISPR is an immune system used by microbes to find and eliminate unwanted invaders.   Qi: CRISPR stands for “clustered interspaced short palindromic repeats.” Biologists use the term to describe the “genetic appearance” of a system, which was first discovered in microbes, including bacteria and archaea, as early as 1987. For a long time, no one really understood what it did, but around 2005, researchers discovered that CRISPR is an immune system. It’s used by microbes to help protect themselves from invading viruses. To stop the invaders, the microbes use CRISPR to recognise and eliminate specific trespassers.

How Does It Work?

CRISPR is a powerful gene-editing technology that allows scientists to precisely modify DNA in living organisms. It works like molecular scissors, using a protein called Cas9 guided by a custom RNA sequence to cut DNA at specific locations. Once the DNA is cut, genes can be removed, altered, or added. CRISPR is widely used in medicine, agriculture, and biological research for treating genetic diseases, improving crops, and studying gene functions.

When a virus or other invader enters a bacterial cell, the bacterium incorporates some of the trespasser’s DNA into its own genome so it can find and eliminate the virus during future infections.   

Qi: It’s similar to the human immune system. When a virus infects us, we generate an immune memory in the form of antibodies – lots of them. Then, when the same virus infects us again, these antibodies quickly recognise the invaders and eliminate them.   

When a virus infects a bacterial cell, CRISPR helps establish a memory – a genetic one. The bacterium takes a piece of the virus’s genome and inserts the DNA into its own genome. From that newly acquired DNA sequence, CRISPR creates a new “guide RNA,” a sequence that helps CRISPR find the invader via sequence complementarity (i.e., A binds to T and C binds to G). So, the next time the virus infects that bacterial cell, the guide RNA rapidly recognises the virus DNA sequence, binds to it, and destroys it.

The History of CRISPR Cell and Gene Therapies

CRISPR cell and gene therapies can treat genetic diseases, cancer, and more. This article explores the history of cell and gene therapies, the CRISPR revolution, and the current state of the field.

Cell and gene therapies have been around for some time, but it was only after the invention of CRISPR that we started seeing more clinical trials bringing in the hope of permanent cures for genetic diseases. In this blog post, we’ll explore the history of cell and gene therapies, the CRISPR revolution, the current state of play, the recent boom in the CRISPR cell and gene therapy field, and the future of these life-changing treatments.

What is Cell and Gene Therapy? 

Cell and gene therapies are a distinct group of medical technologies that aim to treat diseases through the transfer of either whole cells (cell therapy) or genetic material (gene therapy) into patients. While these technologies have some overlap, there are key differences between cell therapies and gene therapies.  

Gene Therapy 

Gene therapy refers to the introduction of new genetic material or the modification of a patient’s faulty genes associated with a particular disease. These modifications may be done directly inside the patient’s body (in vivo) or by editing cells outside of the body (ex vivo). There are over 5,000 known monogenic diseases, meaning that they are caused by mutations in a single gene. In these cases, correcting the mutated gene or delivering a functional copy of the gene can alleviate the condition.  

Cell Therapy 

Cell therapy involves transfusing a patient with healthy cells to compensate for a lack of certain cells or to replace diseased cells. Cell therapy can be either autologous, meaning that the cells used are derived from the patient, or allogeneic, meaning that they are derived from healthy donors. Gene-edited cell therapy involves removing cells from patients and editing them ex vivo to either correct disease-causing mutations or add new genes that will make them more robust for treating disease. Common cell types used in this type of therapy are hematopoietic stem and progenitor cells (HSPCs), induced pluripotent stem cells, and immune cells.

Genetically modified organisms - GMOs      

Genetically modified organisms (GMOs) are plants, animals, or microbes that have had their DNA changed using genetic engineering techniques. Another term for this is bioengineered foods.

Synthetic Biology 

Synthetic biology is a field of science that involves redesigning organisms for useful purposes by engineering them to have new abilities. Synthetic biology researchers and companies around the world are harnessing the power of nature to solve problems in medicine, manufacturing, and agriculture.

Importance: 

The CRISPR/Cas9 technique is one of several gene-editing tools. Many favour the CRISPR/Cas9 technique because of its high degree of flexibility and accuracy in cutting and pasting DNA. One of the reasons for its popularity is that it makes it possible to carry out genetic engineering on an unprecedented scale at a very low cost. How it differs from previous genetic engineering techniques is that it allows for the introduction or removal of more than one gene at a time. 

This makes it possible to manipulate many different genes in a cell line, plant, or animal very quickly, reducing the process from taking a number of years to a matter of weeks. It is also different in that it is not species-specific, so it can be used on organisms previously resistant to genetic engineering.  

The technique is already being explored for a wide number of applications in fields ranging from agriculture to human health. In agriculture, it could help in the design of new grains, roots, and fruits. Within the context of health, it could pave the way to the development of new treatments for rare metabolic disorders and genetic diseases ranging from haemophilia to Huntington's disease. 

It is also being utilised in the creation of transgenic animals to produce organs for transplants into human patients. The technology is also being investigated for gene therapy. Such therapy aims to insert normal genes into the cells of people who suffer from genetic disorders such as cystic fibrosis, haemophilia, or Tay-Sachs. Several start-up companies have been founded to exploit the technology commercially, and large pharmaceutical companies are also exploring its use for drug discovery and development purposes.  

The importance of the CRISPR/Cas9 was recognised with the awarding of the Nobel Prize in Chemistry to Jennifer Doudna and Emmanuel Charpentier on 7th October 2020. What is missed in the awarding of the Prize is the significant role that many others, including Virginijus Siksnys, played in helping to bring about the development of gene editing.

Issues 

In April 2015, a Chinese group reported the first application of CRISPR/Cas9 to (non-viable) human embryos. This development, together with the decreasing costs of the technology, has triggered a major bioethical debate about how far the technology should be used. The technology faces two major issues.  The first issue is a philosophical dilemma. It centres on the extent to which CRISPR/Cas9 should be used to alter 'germ-line' cells - eggs and sperm - which are responsible for passing genes on to the next generation. 

While it will take many more years before the technology will be viable to use to create designer babies, a public debate has already begun on this issue. So great is the fear that some scientists, including some who helped pioneer CRISPR/Cas9, have called for a moratorium on its use in germ-line cells.  

The second issue is one of safety. One of the major problems is that the technology is still in its infancy, and knowledge about the genome remains very limited. Many scientists caution that the technology still needs a lot of work to increase its accuracy and make sure that changes made in one part of the genome do not introduce changes elsewhere, which could have unforeseen consequences. 

This is a particularly important issue when it comes to the use of technology for applications directed towards human health. Another critical issue is that once an organism, such as a plant or insect, is modified, they are difficult to distinguish from the wild-type, and once released into the environment could endanger biodiversity.  

Policy-makers are still debating what limitations to put on the technology. In April 2015, the US National Institutes of Health issued a statement indicating that it would not fund any research that uses genome editing tools such as CRISPR in human embryos. Meanwhile, the UK's Human Fertilisation and Embryology Authority, under whose remit such research would fall, has indicated that the CRISPR/Cas9 technology can be used on human-animal hybrid embryos under 14 days old. Any researcher working in this area would need to first get a license from the Authority. Other leading UK research councils have indicated that they support the continued use of CRISPR/Cas9 and other genome editing tools in preclinical research.  

As regulators debate what restrictions to enforce with CRISPR/Cas9, the technology has become the subject of a major patent dispute. The first application to patent the technology was filed by DuPont in March 2007 (WO/2007/025097). This covers the use of the technology to develop phage-resistant bacterial strains for food production, feeds, cosmetics, personal care products, and veterinary products. Since then, three heavily financed start-up biotechnology companies and half a dozen universities have filed patents. Two major competing patent claims have been filed in the US. The first, filed on 25 May 2015, is grounded in the work led by Jennifer Doudna at the University of California, Berkeley, and Emmanuelle Charpentier, originally at the University of Vienna and now at the Helmholtz Centre for Infectious Research in Germany. The application has 155 claims and covers numerous applications for a variety of cell types (US Patent Application No. PCT/US2013/032589). 

The second was filed by MIT-Harvard Broad Institute on 12 December 2012 for the work of Feng Zhang, which focused on the use of CRISPR/Cas9 for genome editing in eukaryotic cells. It was given fast-track status and was granted on 15 April 2014 (US Patent No. 8,697,359). In April 2015, Charpentier and the Universities of California and Vienna filed a challenge to the patent with the US Patent and Trademark Office. It will take several years for the patent dispute to be settled. The legal wranglings over patents are unlikely to affect the use of CRISPR for basic research because the technology is available through an open-source repository. However, it could have an impact on clinical applications using the technique.  

This scientific profile was written by Lara Marks in June 2016 with generous input from Silvia Camporesi, Xiofan Zeng, and Jonathan Lind. The piece was updated by Lara Marks in October 2020.