Targeted genome editing is a laboratory tool used to remove or insert the DNA of a gene of interest to understand its biological function and relevance. Genome editing has direct application in the clinic, as it is the basis for gene therapy. Gene therapy is a type of treatment where DNA is therapeutically delivered to combat genetic diseases such as cystic fibrosis, muscular dystrophy, sickle-cell anemia, and leukemia. Recently, a huge development has been made in the genome-editing field. This advancement has the potential to treat diseases in a highly efficient and cost effective manner – qualities you do not typically hear when referring to medicine. It has been regarded as “the biggest biotech discovery of this century” and it was discovered in some of the Earth’s tiniest organisms – bacteria.
What is CRISPR and how was it discovered?
The name of this new gene editing technology is CRISPR, which stands for clustered, regularly interspaced, short palindromic repeat. The discovery of these short segments of DNA was actually first published in a 1987 paper in the Journal of Bacteriology, but its wide application has just been recently revealed by Emmanuelle Charpentier (of the Helmholtz Centre for Infection Research and Umea University) and Jennifer Doudna (of the University of California at Berkeley and the Howard Hughes Medical Institute). The discovery was made, like many scientific discoveries, by pure chance.
Doudna and Charpentier, collaborators, were working on elucidating the mechanism by which bacteria defends themselves against viral infection. Their research pursuits were initially basic science-driven without realizing the significant world impact this could have, “One day…we realized, gosh, this could be a very powerful technology,” said Doudna. By performing basic research, they were able to identify a very specific mechanism used by bacteria to defend against viruses and in turn employ this mechanism to larger organisms. In the following paragraphs, we will discuss the mechanism behind CRISPR, its practical and potential applications, and finally how this very powerful tool will be regulated in our society.
How does CRISPR work?
Targeted genome editing relies on the induction of a double stranded break (DSB) in the DNA molecule, which can be accomplished using specific nucleases. Nucleases are enzymes that can break the bonds within DNA. The cleavage of DNA by nucleases can be repaired by one of two mechanisms – non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is the predominant DSB repair mechanism that uses short DNA sequences from single-stranded DNA overhangs on the ends of DSBs. Alternatively; HDR is used when homologous DNA “donor templates” are introduced into the cell, which is then used to repair the DSB. The major difference between the two pathways is that HDR is considered to be more precise and error-free because of the presence of an externally added homologous DNA template, whereas NHEJ does not assure a homologous DNA template present, which may result in a loss of genetic information. This is important because if the DNA sequence is changed after repair, this could lead to mutations and ultimately the production of a different protein.
CRISPRs are short segments of DNA repeats found within many bacterial genomes. Between these repeats are bits of DNA that were found to match viral DNA. This led to the idea that these DNA repeats could be used as an immune defense mechanism by bacteria against viruses so that if the virus attempts to infect the bacteria again, the surviving bacteria can recognize the viral DNA by storing its genetic information within these DNA repeats, kind of like a mug shot. Once the viral DNA has been incorporated into the host genome between these CRISPRs, it is transcribed and processed to produce small RNAs known as CRISPR RNA (crRNA). These crRNAs are very important because they contain complementary regions to that of the viral DNA, which is then used to guide the nucleases to the foreign site. Now, if the viral perpetrator returns, CRISPRs can harness their power of memory to order an attack against the virus. With this order, crRNAs are released into the cell, which act as a road map to guide the nucleases to the viral DNA. These nucleases are the soldiers that drive this molecular attack by chopping up the viral DNA. After this happens, the cell uses one of its repair mechanisms (described earlier as NHEJ or HDR) to patch up the wound.
What can we use CRISPR for?
Now how could a system used in bacteria be applied to humans? By understanding the mechanism of CRISPR in bacteria, scientists can recapitulate the same system artificially in a laboratory and apply it to their organism of choice. The concept is that the guide sequence within the crRNAs can be engineered to contain a sequence of interest, which will then reorient the specific nucleases to your gene of interest. In the lab, scientists are able to provide the donor DNA template and induce the HDR pathway, ensuring precise and error-free DNA repair. This technique can be performed relatively easily in virtually all living organisms and at a much lower cost, distinguishing CRISPR as a highly efficient genome editing technology.
Thus far, the CRISPR system has been successful in worms, zebrafish, fruit flies, mice, rats, and a number of plant species. CRISPR is becoming an increasingly routine practice in the world of agriculture. For instance, introduction of mutations or other genetic changes into plants can enhance breeding of certain crops such as rice and wheat. But CRISPR can work beyond improving crops; it has the potential to manipulate mammalian genomes for therapeutic purposes.
A group of Chinese scientists recently published the use of CRISPR in non-viable human embryos to study a fatal genetic blood disease called β-thalassaemia. These embryos came from a fertilization clinic but were not used because they had severe genetic abnormalities that would prevent a live birth from occurring. However, these embryos still go through the earlier stages of development. This group sought out to delete a single gene in human embryos that is responsible for β-thalassaemia. They allowed the embryo to go through a few cell divisions before collecting the samples for genetic analysis. They genetically tested over 50 different embryos and half of them showed that the gene was successfully deleted by CRISPR – an inefficient rate but a promising start. The authors also reported instances of “off-target” or random mutations in some of those treated embryos. This shows us that although CRISPR does have a bright future, it is still in its rudimentary phase in regard to clinical application. However, there are still some critiques concerning the merit of this study. For example, the somewhat disappointing initial results may be due to the defects of these embryos and may not represent how CRISPR will function in normal embryos. Further optimization of CRISPR needs to be done to be a suitable technique for treating human diseases.
What are the ethical concerns regarding CRISPR?
In the last 2 years, funding for CRISPR related projects has skyrocketed. With its increasing use, there are also concerns regarding the ethics of CRISPR studies and how it should be used. The aforementioned study using non-viable human embryos was still subject to debate due to its ethical ambiguity. Due to these ethical concerns, the publication was initially rejected to top tier journals such as Nature and Science, which are typically known for their groundbreaking content.
The rise of CRISPR has ignited intense debate on whether we should be using this technology to induce heritable alterations in a person’s genome. Given the extensive knowledge we have of the human genome, scientists are able to pin point specific genes that cause certain diseases, like β-thalassaemia and leukemia. However, Doudna herself expressed that we should not be using CRISPR on humans that have not been born yet. She explained recently on a radio interview, “That is a permanent change, that is a change to the DNA that will be passed onto to their children…and you can’t ask the person if that’s okay because you are doing it before they are born.”
In March, Doudna along with a group of leading biologists, including Nobel laureate David Baltimore, published a letter in the journal Science to initiate a public discussion about the safety and ethical concerns of CRISPR and to request a moratorium in regards to human genome editing via CRISPR. But some scientists think a moratorium is unnecessary and that we should continue to push CRISPR forward as a potential therapy. Henry Miller, a medical researcher, also published a letter to Science recently where he asks, “Shouldn’t 21st-century medicine offer the possibility of repairing embryos that will become patients [for example] with sickle cell disease, and eliminate the disease from future generations?” Miller and others feel that although we should lead with caution when it comes to using CRISPR for clinical research, instating a moratorium is insensitive to those patients currently suffering from diseases that may benefit medically from this new gene editing technology.
It will be challenging to move forward with CRISPR as a therapeutic strategy in humans if we are not able to test this on viable human embryos. We have seen many incredible medical advances in our lifetime that were once subject to intense ethical scrutinty – just look at in vitro fertilization (IVF). People were inherently skeptical about the practice of making babies in a laboratory setting because it was considered “unnatural”. However, today around 60,000 babies are born each year via IVF. Creating a public dialogue concerning CRISPR is an important first step that will hopefully open the doors to new and effective therapies while still maintaining trust between scientists and the general public.
Editor: Kimberly Maxfield