Since CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing was discovered in 2012, the technology has garnered a lot of attention and generated significant excitement for its ability to make precise, permanent changes to DNA in animals and plants.
By altering genetic sequences, CRISPR has the potential to provide novel therapies for patients suffering from severe diseases caused by a single gene mutation, including sickle cell disease (SCD), cystic fibrosis and Huntington’s Disease. CRISPR is also being investigated as a treatment for acquired immune deficiency syndrome (AIDS) and to improve anti-tumor immunotherapy. Its use will no doubt expand beyond these disease areas as the technology evolves. CRISPR gene editing may also be used to improve crop resilience, improve yields and boost nutritional value to help feed the growing world population with the finite resources available.
With constant conversations examining the potential of the technology, it can be hard to focus on what has been achieved since 2012 and to look toward the future to determine what may be achievable both in the near – and longer – term.
How far we have come
While CRISPR gene editing is still in its infancy, with many unknowns and questions remaining, we are already seeing the first therapeutic applications being tested in human clinical trials. In 2018, a cancer immunotherapy clinical trial was opened at the University of Pennsylvania. In this trial, CRISPR genome editing is used to modify the patient’s own T-cells to express an ESO-1 chimeric antigen receptor and eliminate PD-1 expression.
In 2019, Vertex Pharmaceuticals and CRISPR Therapeutics opened clinical trials to treat b-thalassemia and sickle cell disease by using genome editing to re-activate expression of fetal hemoglobin in the patient’s own hematopoietic stem cells, functionally replacing the defective b-hemoglobin gene that causes these disorders. This trial will run in the US, Canada, and Europe.
Both of these trials involve CRISPR editing performed “ex-vivo”, wherein patient stem cells are removed, genome editing is performed in a laboratory and the modified cells are later reinfused to the patient as the active therapy. An in vivo human clinical trial was opened by Editas Medicine and Allergan in 2019 to treat Leber congenital amaurosis 10 (LCA10), an inherited form of blindness, by direct subretinal injections of a CRISPR AAV viral vector.
Although these trials represent the first use of CRISPR methods to treat human diseases in the US, the first CRISPR clinical trials were actually performed in China, and nine trials are currently listed as actively recruiting patients in China on the US Government Clinical Trials database.
These studies will take years to run and analyze results. Throughout the process, a key focus will be the monitoring of any off-target effects (OTEs) in these patients. CRISPR gene editing relies on pre-programmed nucleases that target specific sequences in the genome and then introduce cuts into the DNA strands. These cuts allow for the removal of existing DNA and replacement with modified DNA. However, cleavage and editing may occur at additional sites with similar sequences to those found at the intended site. In addition, even targeting the right sequence can produce unintended effects since we still do not have a full understanding of the function of each gene.
Therefore, there remains a need to continue to better predict and control OTEs, and one way to do so is by improving the specificity of the tools used in CRISPR gene editing. This is a focus of our work at Integrated DNA Technologies (IDT), and we have developed a new Cas9 enzyme that improves targeting specificity and reduces OTEs compared with the wild type (WT) enzyme. To do so, we created an unbiased bacterial screen to isolate Cas9 variants that provide highly specific cleavage with minimal OTEs, while keeping the nuclease activity comparable with that of WT Cas9. The results of this work and clinical utility were published in Nature Medicine in 2018.1 As the most active and specific high-fidelity Cas9 variant available when delivered as a ribonucleoprotein (RNP) complex, HiFi Cas9 is ideal for use in clinical studies.
As clinical studies of gene therapies continue to generate interest in the mainstream media and among lay audiences, it will be important to improve the understanding of what CRISPR is and the potential it offers, as well as clarify the difference between germline and somatic gene editing to prevent misinterpretations. If the pace of scientific progress moves faster than the pace of consumer awareness and understanding, there is a risk that the technology will face public rejection, as is sometimes seen today for the introduction of genetically modified organisms (GMOs) in agriculture.
Beyond human disease
When thinking about the potential for gene editing, we must not limit ourselves to discussions revolving around human disease. Even greater, more widespread societal benefits might be more quickly realized through appropriate agricultural use of these tools. While Cas9 remains the best-characterized and most widely used nuclease for mammalian gene editing, Cas12a has recently emerged as an alternative and is already gaining widespread acceptance in agricultural science. There are several unique features of Cas12a that distinguish it from Cas9, most notably the fact that it targets AT-rich regions of the genome, which makes it ideal for editing certain plants, which are AT-rich. Until recently however, Cas12a was not very efficient at cutting DNA. However, our team at IDT engineered a new and improved variant, Cas12a Ultra, which not only provides specificity and efficiency as good as that of Cas9 but also works across a broad range of temperatures.
Research is underway to determine CRISPR gene editing’s potential to improve food-related outcomes, including developing soybeans lower in unhealthy fats, bolstering the cacao plant’s ability to defend itself against a virus and developing tomato plants that produce more tomatoes.2 In the future, we may also start to see CRISPR improving crop efficiencies by developing corn that is able to withstand droughts or vegetables that have been fortified with nutrients, as well as greater access to gene edited foods since it is simpler, safer, cheaper and quicker than historical methods used to produce the first generation of genetically modified organisms (GMOs). As such, we may well find CRISPR-engineered foods available in markets before CRISPR-based therapies are approved and available.
A look to the future
While several exciting clinical trials are already open, we can expect that even more will be announced in 2020 and subsequent years. The focus of these early trials will be treatment of monogenic disorders, where a clear gene target exists that can be modified to alter disease progression. While final results will take years to unfold, there will no doubt be significant learning from interim points in the studies as well as from personal stories as patients begin to see the impact of their treatment. While a long-term goal for the research community will be to evolve CRISPR therapies beyond diseases caused by single gene mutations, such advances will come more slowly as we learn how to reduce risks associated with performing editing on multiple genes simultaneously.
The potential of gene editing cannot be ignored, but there are still many hurdles, both scientific and societal, that must be overcome. The societal hurdles, which are no less significant, result from the rapid pace of scientific change and require consumer understanding and knowledge to keep pace. Ensuring that publicly available information is accurate, accessible and easy to understand will be critical to increase the scientific literacy of the general public and support scientific progress. These hurdles will require the entire scientific community, and beyond, to come together to overcome but could lead to one of the greatest scientific revolutions in recent times.
1. Vakulskas CA, Dever DP, Rettig GR, Turk R, et al. (2018) A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nature Medicine 24, 1216–1224.
2. Niler, E. (2018) Why Gene Editing Is the Next Food Revolution. National Geographic.