Genome Editing: Is CRISPR/Cas the Answer?

by Jackson Allen

For roughly the last 60 years, the focus of molecular biology and genetics has been to better understand the microscopic machinery that regulates the genome of every organism. Such scientists as Matthew Meselson, Rosalind Franklin, James Watson, and Francis Crick helped pave the way for contemporary understanding of molecular genetics. These scientists were responsible for discoveries including the structure of DNA, the molecule that encodes all of the genetic information of a person. Though the field of molecular biology has grown immeasurably since then, the most important discoveries in the field remain those that elucidate the mechanisms used to regulate genetic information in living organisms.

Unsurprisingly, scientists have begun to ask how they could apply this knowledge to conquer disease, correct genetic problems, or even learn more about genetics itself. The list of developments in molecular genetics is seemingly endless: genetic therapies promise to reprogram the body’s defenses to target cancer, analysis of the human genome has given us unprecedented understanding of our own genetics, and new technology can simulate the folding of proteins to assist in the development of new pharmaceuticals. However, no genetic tool today seems to hold more power and promise than CRISPR/Cas genome editing technology.

What is CRISPR/Cas?

First observed in 1987, the CRISPR/Cas system in nature comprises the immune system of many bacteria and archaea (1). These single-celled organisms use short repeats of DNA called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) to mark viral DNA that has been incorporated into their genome by Cas (CRISPR Associated) proteins. By incorporating the DNA sequence of attacking viruses into their own genome, these bacteria can destroy an attacking virus by cleaving its DNA with a Cas protein (2). The discovery that bacteria can easily and accurately modify their own genomes remained largely underutilized until the early 2000s, when scientists began to modify viral-resistant bacteria using spacer DNA similar to CRISPRs (3). In 2012, scientists demonstrated that, using CRISPRs and Cas9 proteins, human cells could be genetically modified with precision not seen with other genome editing methods (4). Since then, researchers have used the CRISPR/Cas9 system to modify organisms including zebrafish, plants, and mice. Last year, researchers at the Koch Institute at MIT demonstrated that CRISPR/Cas9 could be used to cure mice of a genetic disease that prevents the breakdown of the amino acid tyrosine and eventually causes liver failure. After an injection of CRISPR RNA and Cas9 paired with DNA for an enzyme that breaks down tyrosine, the mice began to produce this enzyme. Within 30 days, the mice were cured of the disease and no longer required daily medications (5).


The CRISPR/Cas9 system can be used for gene silencing as well as gene modification. Both outcomes have the potential to make important contributions to laboratory research and disease treatment. The CRISPR/Cas9 system relies on two main components: a Cas9 endonuclease that can cut DNA in the nucleus of a cell and a guide RNA, made of CRISPR RNA and trans-activating CRISPR RNA (tracrRNA). In nature, the CRISPR and tracrRNAs are separated. However, researchers discovered that the two sequences could be combined into a single guide RNA, significantly reducing the complexity of the system (6). The CRISPR RNA directs the Cas9 endonuclease to the appropriate DNA cleavage site, while the tracrRNA stabilizes and activates the Cas9 endonuclease. When this protein is activated, it creates a double-stranded break in the target DNA, which leads to activation of cellular repair mechanisms (6). Double-stranded DNA repair often leads to random insertions or deletions in the gene, because neither strand can serve as a template for repair. These mutations often silence the affected gene and prevent binding of the guide RNA used to target the gene. Thus, the CRISPR/Cas9 system will continue to target the DNA until such a mutation is introduced. However, if a segment of single-stranded DNA that is complimentary to either strand of cleaved DNA is introduced, the cell will repair the DNA cleavage using the single-stranded DNA as a template. Scientists have demonstrated that certain genetic mutations can be corrected by introducing this single-stranded DNA template to the cell’s own repair mechanisms (6).

In addition, the use of Cas9 nickase, a specialized version of the Cas9 endonuclease, has been shown to only cleave one strand of a cell’s DNA, reducing the frequency of off-target modifications while still allowing for DNA repair from a single-stranded DNA template (7). This specificity makes the CRISPR/Cas9 system more accurate and less likely to edit DNA at an undesired location. In fact, two studies in 2013 demonstrated that off-target modifications were reduced by 50 to 1500 times when Cas9 nickase was used (8).

However, treatments using CRISPR/Cas9 may not be limited to genetic diseases. A study at the Whitehead Institute used CRISPR/Cas9 to systematically analyze the effects of silencing over 7000 different genes on resistance to chemotherapy in cancer cells (9). Finding genes that are essential to the survival of tumor cells could potentially lead to a treatment using CRISPR/Cas9 alone or in combination with other therapies. Targeted delivery of CRISPR/Cas9 therapy is also a possibility, although more difficult than IV injections that have been used in previous animal studies. Other scientists working in developmental medicine have used CRSIPRs to screen mouse embryos for genes that could provide resistance to bacterial toxins (10). More effective at silencing genes than RNA interference, screening studies using CRISPR often report gene candidates that would have gone unnoticed in other types of screens (9).

Harvard Medical School Professor of Genetics George Church is one of the leaders in a field of scientists working to expand our knowledge about the CRISPR/Cas9 system. Church’s start-up, Editas Medicine, hopes to develop real-world treatments for genetic diseases using the most recent developments in CRISPR genomic editing. Church points out that the advantages of the CRISPR/Cas9 system over other types of gene editing are crucial for the practicality of treatments based on this science (11). The CRISPR/Cas9 system avoids the problems encountered in other methods of genome editing. For example, viral-vector delivered gene therapy can leave a dysfunctional gene in place even when inserting a healthy gene (11). By contrast, the CRISPR/Cas method is focused on the correction of genes already in an organism’s genome. This approach has the added benefit of leaving the gene in its correct chromosomal location, meaning the cell retains the ability to regulate the gene (11). “Editas is poised to bring genome editing to fruition as a new therapeutic modality, essentially debugging errors in the human software that cause disease,” said Editas director, Alex Borisy, in a recent interview with the McGovern Institute for Brain Research at MIT (11).

To be sure, the successes of CRISPR/Cas9 have not gone without scrutiny in the scientific community. Though most researchers working in the field would support the use of the CRISPR/Cas 9 system for curing genetic diseases like cystic fibrosis or sickle-cell anemia, far fewer support the use of this technology for other genetic modifications like cosmetic changes. Even among those who back the use of CRISPR/Cas9 for targeting disease, the possibility of editing the human germline, cells with the potential to pass on their genetic information, has sparked a debate about the role humans should play in our own evolution. On March 19, 2015, a number of scientists, policymakers, and experts in law and ethics published an editorial in Science, calling for a moratorium on germline genome modification until the ethics of such modifications can be debated openly from a multi-disciplinary perspective (12). This group, which included one of the co-discoverers of CRISPRs, cited a need for greater transparency in discussions of the CRISPR/Cas 9 system. The editorial, resulting from a January 2015 Innovative Genomics Initiative conference on genome editing, also sought more standardized benchmarking and evaluation of off-target modifications made by the CRISPR/Cas 9 system, the effects of which remain largely unknown (12). However, these scientists were careful to point out the tremendous potential of CRISPR/Cas 9 for curing genetic disease, which might tip the balance between risk and reward in favor of responsible genome editing (12).

The Future of CRISPR/Cas

The accuracy, efficiency, and cost of CRISPR/Cas9 make it an attractive alternative to other methods of genome modification. Though they have decades of research behind them, tools such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) still prove costly and complex to use. For example, CRISPR recognition of target DNA depends not on a large and complex protein that must be synthesized, but on simple Watson-Crick base pairing between target DNA and simple RNA (9). Additionally, CRISPR/Cas9 can modify several genes at once (9). For these reasons, CRISPR/Cas9 may find its greatest niche in improving ongoing laboratory research, including the generation of genetically modified animals and cell lines. Other researchers prefer to work outside of the realm of genetic diseases, finding applications for CRISPR/Cas9 in agriculture, ecology, and even the preservation of endangered species. Though the technology behind CRISPR/Cas9 is still young, scientists and start-ups alike have begun to pioneer applications—from pathogen, heat, and drought-resistant crops to the recreation of the wooly mammoth and other extinct species (9, 13).

The coming years promise to be some of the most exciting for molecular biology. Although CRSIPR/Cas9 may just be an application of the knowledge acquired by scientists in recent decades, its promises are truly groundbreaking. With potential uses from genetic diseases to the revival of extinct species, CRISPR/Cas9 could very well usher in a new age of applied molecular genetics. However, this paradigm shift in such a pioneering field of science will not be without its ethical questions and debates. The greatest struggle for science in the future may not be the capabilities of developing technology, but the restraint and responsibility necessary to use such powerful tools. For the first time, quick and efficient editing of an organism’s genome is a realistic possibility, giving humanity the power to control its own evolution at the genetic level—not to mention the ability to change the genetics of the animals and plants that inhabit our world. For the scientists developing this technology, the policymakers calling for open discussions of its ethical issues, and the millions of people who could benefit from it, the coming years will undoubtedly hold tremendous advances.

Jackson Allen ’18 is a freshman, planning to concentrate in Molecular and Cellular Biology.


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Genome Editing: Is CRISPR/Cas the Answer?
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