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The idea of editing genes to treat disease dates back to at least the 1950’s and the discovery of the DNA double helix. When researchers uncovered that genetic information in the form of DNA is passed from parents to children and that small changes in the sequence can be the difference between health and disease, scientists tasked themselves to identify the precise “molecular mistakes” that cause different genetic diseases with the long term goal that ultimately such errors could be fixed to cure the disease in question.
The idea of editing genes to treat disease dates back to at least the 1950’s and the discovery of the DNA double helix. When researchers uncovered that genetic information in the form of DNA is passed from parents to children and that small changes in the sequence can be the difference between health and disease, scientists tasked themselves to identify the precise “molecular mistakes” that cause different genetic diseases with the long term goal that ultimately such errors could be fixed to cure the disease in question.1
The first attempts at “gene therapy” involved introducing a functional copy of the disease gene into the appropriate tissue, hoping that partially replacing normal gene function this way would either cure or at least reduce the negative effects of the inherited gene error. More recently, strategies have expanded to attempt “gene surgery” to repair the DNA coding errors by converting mutations to the correct, normal sequence. Gene surgery requires the availability of enzymes that can target and cut DNA at very specific sites within living human cells as the first step of this repair process. In the case of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), the gene-cleavage technology comprises two components: a nuclease (e.g., Cas9), which acts like a pair of scissors and is responsible for cleavage of double-stranded DNA, and a single guide RNA (sgRNA), which forms a complex with the nuclease and provides sequence specificity, essentially “guiding” the Cas9 nuclease to a single target site within the three billion bases of DNA present in a human genome.
Since CRISPR genome 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. From curing previously untreatable disease to helping feed a growing population, the potential is practically limitless. With all the excitement around the technology, it is important to remember what has already been achieved to-date and speculate what may be possible both in the near- and long-term.
How we got here
In the early 1990s, a scientist completing his PhD in Spain discovered genetic sequences in archaea that were repeated up to 600 times in a row. This paralleled a similar discovery in bacteria by Japanese scientists in the late 1980s. These repeats were later found to be part of a prokaryotic immune system. They are used to store genetic information on bacterial viruses (bacteriophages) to which the organism and its ancestors have been exposed and prime the organism to defend itself against that invader in the future.2 At the time, few people would have believed that this finding, which later became known as CRISPR, would lead to the frenzy in mammalian genome editing we see today.
Other methods of genome editing existed before CRISPR, such as Meganucleases, zinc finger nucleases (ZFNs) and TALENs. However, publications from the Doudna and Zhang groups in 2012-2013 revealed a new system that was faster, cheaper, and easier to perform than the older technologies.3,4 From there, the CRISPR era began. Unfortunately, CRISPR technology is not perfect. DNA cleavage can occur at unintended sites that are similar but not identical to the desired target site. These are known as off-target effects (OTEs) and in some cases may account for more than 50% of the editing delivered by wild type (WT) Cas9.5
Challenges with CRISPR-Cas9 genome editing
While CRISPR/Cas9 genome editing offers unparalleled gene editing efficiency in various cell types and species, challenges remain relating to target site specificity. Cutting DNA at the precise site needed to correct a genetic error is the intended outcome. However, sometimes the Cas9 nuclease cuts at sites that are similar but not identical to the sgRNA, which may differ by one to four bases. While sometimes these sites may lie in area of “junk” DNA, there is always a risk that an important gene function or regulatory element might be impacted, making off-target editing a potentially significant risk for human therapy.
Numerous attempts have been made by scientists to improve the specificity of the Cas9 enzyme, including our team at Integrated DNA Technologies (IDT). By devising an unbiased bacterial mutagenesis screen to isolate Cas9 variants, we were able to develop a high-fidelity Cas9 enzyme, known as HiFi Cas9. HiFi Cas9 offers highly specific cleavage with minimal OTEs while keeping the desired on-target nuclease activity in line with that of the wild type (WT) Cas9. The results of this work were highlighted through the application of HiFi Cas9 in human stem cells, published in Nature Medicine in 2018.6 IDT has partnered with Aldevron to supply a Good Manufacturing Practice (GMP) grade of the enzyme for clinical use. This enzyme is currently being used in clinical trials of investigational CRISPR therapies.
Using CRISPR genome editing to cure disease
By altering gene sequence, 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.
While still in its infancy, with many unknowns and questions remaining, we are already seeing the first therapeutic applications being tested in human clinical trials. CRISPR is currently being evaluated in early phase clinical trials for several disorders caused by a single gene mutation including SCD and β-thalassemia. Many of the early gene editing trials will be done “ex vivo”, meaning that patient cells are removed from the body, treated, then re-infused. This avoids the risks associated with injecting a therapy into a person that can permanently alter their DNA. Given the progress made in a relatively short timeframe, I believe we will be hearing a lot more about CRISPR in 2020 and the years to come.
As we build our understanding of the technology, and fully understand the opportunities it may afford as well as begin to see the outcomes of early clinical trials, I anticipate that we will begin to broaden use from diseases caused by single-DNA base mutations to more complex diseases caused by multiple-base mutations, possibly targeting correction of more than one gene simultaneously. Furthermore, we will increasingly see applications developed for invivo rather than ex vivo use. These invivo therapies will start out as highly targeted and localized treatments, for example, therapies delivered to a specific site like the eye (an ocular CRISPR clinical trial is already in progress) but could eventually involve systemic somatic invivo treatments. This will, of course, require many years of testing but it is anticipated that inroads will be made towards this goal in this decade.
We will also likely witness the arrival and application of new technologies similar to the predominant CRISPR/Cas9 system. Following a presentation at the Cold Spring Harbor Laboratory in October 2019, there was much excitement about the potential of “PRIME editing.” While this new variant genome editing technology is exciting, it is still in its infancy, compared with current conventional CRISPR technology. It will be important to evaluate its strengths and weaknesses and assess any OTEs. Similarly, base editing, another newer genome editing approach, is likely to continue to be investigated, for introducing specific point mutations into DNA or RNA without the need for double-strand breaks. Such interventions can conceivably also be used to change the sequence or abundance of RNA transcripts, as well as alter DNA sequences.7 No doubt there will be other discoveries that will show potential, but it remains to be seen which ones will lead to eventual therapies and other real-world applications. Such successes will likely also go on to inform the ongoing debate regarding germline genome editing.
Applications beyond human disease
When thinking about the potential for gene editing, we must not limit ourselves to only thinking about 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 a second, important option in the genome editing toolbox 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 broader range of temperatures, which can be critical in certain agricultural applications.
Research is underway that employs CRISPR to improve our food supply, 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.8 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, cheaper, quicker and more precise 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 medical therapies are approved and available.
Learning from past mistakes
While there is currently much excitement about CRISPR gene editing, particularly among people who suffer from genetic diseases that are currently untreatable, the question remains: how will we maintain public support as the technology evolves? CRISPR and other gene editing therapies offer patients suffering debilitating diseases hope that someday there will be a curative therapy for them. However, as we have seen with other scientific advancements, it will be critical for patient/consumer awareness and education to keep pace with scientific progress to ensure people are well informed about CRISPR’s potential and confident that enough regulatory oversight is being applied. This will require a concerted effort and open dialogue across society including scientists, academics, the media, policymakers, patients and other consumers.
1. Fridovich-Keil JL. Gene Editing. Encyclopedia Britannica, Inc. June 4, 2019. https://www.britannica.com/science/gene-editing. Accessed December 2019.
2. Ishino Y, Krupovic M, & Forterre P. History of CRISPR-Cas from encounter with a mysterious repeated sequence to genome editing technology. Journal of Bacteriology. 2018; 200: e00580-17.
3. Jinek M et al. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012;337: 816.
4. Le Cong et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. 2013;339: 819.
5. Zhang, X. H., Tee, L. Y., Wang, X. G., Huang, Q. S., & Yang, S. H. Off-target effects in CRISPR/Cas9-mediated genome engineering. Mol. Ther. Nucleic. Acids. 2015;17: 4.
6. Vakulskas CA, Dever DP, Rettig GR, Turk R, et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat Med. 2018;24:1216–24.
7. Rees HA, Liu DR. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet. 2018;19: 770–788.
8. Niler, E. Why Gene Editing Is the Next Food Revolution. National Geographic. 2018.
Mark Behlke, chief scientific officer at Integrated DNA Technologies (IDT)