The CRISPR-Cas9 revolution

Author:  Maria Zagorulya

Institution:  University of Rochester

The discovery of the DNA double helix in the mid-twentieth century heralded a revolution in modern biology. The newfound understanding of how hereditary information is passed down set the foundation for the development of tools to modify and manipulate genes in ways only nature had over billions of years of evolution. Today, the fruits of genetic engineering are widespread: laundry detergent with genetically modified enzymes allow for a more effective wash; golden rice with increased amounts of vitamin A prevents nutritional deficiency; and bacterially synthesized human insulin treats patients with type I diabetes. Recently, a new set of tools for genetic engineering—the CRISPR-Cas9 system—has taken the scientific and public world by storm. But in addition to new hopes about tackling genetic disease, questions and concerns have emerged about the ethics of genetic engineering, a controversy that has mired over this field of research for almost as long as it has existed.

Genetic engineering involves the use of modern biotechnology to add, remove, or change portions of an organism’s genome, as well as transferring genes from one organism to another. If performed in embryonic stem cells, such precision DNA modifications can lead to the creation of an entire organism with an altered genetic makeup. An important application would be the ability to correct genetic mutations at the level of an embryo, allowing for the development of a healthy organism. In 2012, the discovery of the CRISPR-Cas9 system in bacterial immunity by the collaboration of the Emmanuelle Charpentier lab at Umeå University in Sweden and the Jennifer A. Doudna lab at the University of California, Berkeley has brought us remarkably close to the development of a cure for genetic diseases.

The CRISPR-Cas9 technology has been optimized considerably since its development, but the basic biological concepts remain the same. The assay consists of two units: the Cas9 enzyme, which cuts the DNA double helix, and a guide-RNA, which directs Cas9 to specific sites in the genome. If a cut is made in a gene, DNA repair machinery in the cell silences the gene. By introducing a new DNA fragment with the Cas9-gRNA complex, the DNA repair machinery repairs the DNA by incorporating the new DNA where the cut was made. This way a specific mutation can be introduced into a gene, or a mutated gene can be replaced with a healthy one, or an entirely new gene can even be introduced into the cell DNA. Essentially, CRISPR-Cas9 allows scientists to manipulate cell DNA in an infinite number of ways.

Since its discovery in bacteria, the CRISPR-Cas9 system has been shown by a number of researchers to function in various other organisms, and has been a major scientific breakthrough. Previous genetic engineering methods have been significantly more time-consuming, labor-intensive and limiting, a contrast to CRISPR-Cas9, which allows for simple, efficient, quick and flexible gene editing. The CRISPR-Cas9 tool has gained popularity among biological researchers, as its simplicity allows for its use by all life scientists, including non-specialists, for genetic engineering. Since the development of the technology, the number of patents and publications utilizing the CRISPR-Cas9 tool has increased dramatically, a trend that is only expected to continue as the technology is further refined.

Scientific interest in CRISPR-Cas9 has soared mainly due to increased opportunities to study different genes and flexibility of application. By cutting out or modifying a gene of interest, scientists can directly study the function of both healthy and mutated forms of the gene. Furthermore, flexibility makes CRISPR-Cas9 a suitable method to study any gene, regardless of its location or DNA composition. This flexibility stems from the guide-RNA design: scientists can easily create a guide-RNA to target any site in the entirety of an organism’s genome. However, while CRISPR-Cas9 has been a convenient research tool, improvements and further understanding of the technology are needed before its application for clinical use.

Until recently, the biggest issue preventing the use of CRISPR-Cas9 to edit human genes has been off-target effects. Off-target effects refer to the ability of Cas9 to sometimes cut DNA at the wrong site. This is not a big issue in the use of the tool for research applications, because gene editing experiments are performed on many cells, and of these only the cells with the correct DNA changes will be selected for further study. In order for the technology to be considered for therapeutic use, however, it needs to be very precise. The potential of Cas9 to cut in non-targeted locations results in the possibility of undesired gene edits, which will alter or even silence functions of non-targeted genes, causing various health problems and diseases including cancer.

A partial solution to the CRISPR-Cas9 precision problem was proposed in 2013 by the Feng Zhang lab at the Broad Institute of MIT and Harvard; the research team modified the original wild-type Cas9 enzyme to cut only one DNA strand (the mutated enzyme is called a nickase). Scientists found that using a Cas9 nickase, as opposed to wild-type Cas9, resulted in fewer off-target effects. While this was an improvement, even higher precision was required for potential therapeutic use.

In November 2015, the Zhang lab created a new modified Cas9 enzyme, called eSpCas9, the off-target effects of which were undetectably low. The scientists studied the structure of wild-type Cas9 and its interaction with DNA, and used this knowledge to decrease the off-target effects of Cas9.  They changed three amino acids in the composition of wild-type Cas9, and the modification resulted in incredibly high precision, one that may in fact be good enough to be used for gene editing in humans.

The therapeutic potential of CRISPR-Cas9 is certainly at the forefront of scientific interest and investigations. Studies have shown the ability of CRISPR-Cas9 to correct mutations underlying a number of genetic diseases including cataractsmuscular dystrophybeta-thalassemia, and hereditary tyrosinemia in mice.

In April 2015, the Junjiu Huang lab at the Sun Yat-sen University in Guangzhou published the first-ever study of CRISPR-Cas9 gene editing in non-viable human embryos. The results showed low efficiency and a high number of off-target effects, and the researchers concluded that the CRISPR-Cas9 technology is not yet precise enough for therapeutic use in humans. Perhaps eSpCas9 will substantially reduce the number of off-target effects, but such studies have not yet been done. Thus, more optimization may be needed before scientists and clinicians become confident in using the technology in humans. However, the anticipation of the application of the technology in human therapy raises ethical and social concerns, which must be addressed.

The rapid developments in CRISPR-Cas9 genetic engineering technology prompted the International Summit on Gene Editing in early December 2015 to discuss the ethical, social and legal issues on the use of the technology for gene editing in humans. The summit was held by the US National Academies of Sciences and Medicine, the UK Royal Society, and the Chinese Academy of Sciences. There was a unanimous agreement that the study of gene editing to correct defects after birth should continue. However, no agreement was reached on gene editing in the human embryo, although these experiments were not condemned. The necessity to resolve relevant safety and efficacy issues prior to performance of gene editing in human embryos was highlighted.

It is not long until research will advance the knowledge of using CRISPR-Cas9 in human embryos, but there is no social consensus on the matter. Many fear that the new technology will give rise to “designer babies”, genetically modified children designed to contain traits of intelligence, beauty and/or health. A related issue, raised at the summit, is the potential of genetic engineering to increase societal inequality, as rich people will be able to choose physical characteristics for their children. However, others argue that if a cure is developed, scientists and clinicians will have a moral obligation to help people with debilitating genetic diseases, including babies to be born with severe genetic defects. Another popular opinion is that of the inevitability of human gene editing, given the simplicity and global accessibility of the CRISPR-Cas9 technology.

Perhaps, all these opinions can be best reconciled with implementation of strict regulations to control the use of genetic engineering. A potential solution is that effective regulation will ensure the technology is used only for disease cure and prevention, and thus allow genetic engineering to continue to change human lives for the better.

Until then, over the course of 2016, a multidisciplinary committee of experts will be performing a comprehensive review of the science and policy of human gene editing. A consensus report detailing the official view of the National Academies of Science and Medicine is scheduled for release late in 2016. This report will offer guidelines on the ethical limits and opportunities in human gene editing, and evaluate the possibility of its use in therapy.



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