According to doctors, Vincent Freeman was born with faulty genetics. His parents were notified that he had a high probability of developing a variety of disorders and, at best, would not live beyond his mid-30s. From an early age he “came to think of [himself] as others thought of [him]—chronically ill. Every skinned knee and runny nose was treated as if it were life-threatening.” Two years after Vincent was born, his parents decided to have a second child, Anton. Unlike his older brother, Anton was genetically superior: “a son [his] father considered worthy of his name.”
Anton’s genome was superior because his parents consulted a geneticist before in vitro fertilization. Together, they selected what traits Anton should and shouldn’t have. They wanted blue eyes, dark hair, and fair skin and no propensity for obesity, alcoholism, violence, or any possible inherited genetic disorder.
At this point, you may be suspicious of the validity of this story where parents can choose their child’s traits. And your skepticism is warranted; this story is in fact the plot for the 1997 film Gattaca, starring Ethan Hawke. Hawke, who plays Vincent, grows up healthy and becomes an astronaut despite being born with unfavorable genetics and living in a society ruled by genetic discrimination.
Thankfully, we do not live in that society. For one, Congress passed the Genetic Information Nondiscrimination Act in 2008, which specifies that your genetics cannot be used as a reason for denied health insurance, higher premiums, or denied employment. It is comforting that we have some restrictions preventing the abuse of our genetic information. However, where are we in terms of selecting our children’s genetics? Could Vincent’s genetic “faults” be fixed? There are profound ethical and philosophical issues that are inherent to whether we should or shouldn’t do it. But can we do it?
How we edit genes
Studies in the early 1990s in baker’s yeast showed that we could change genes by physically cutting them and providing them with a novel template for repair. Researchers used a pair of molecular scissors — a homing endonuclease enzyme called I-SceI — that identifies and cuts at a short, unique sequence in the genome. This sort of break in the genome is highly detrimental, and yeast encode a robust mechanism to fix it. By co-opting this machinery, however, researchers could add, delete, or modify genes. Like yeast, human cells contain similar DNA repair machinery. In fact, all other living organisms on our planet have it as well. Therefore, this method of gene editing can work in all species.
From left to right: Twitter CEO Dick Costolo, Emmanuelle Charpentier, PhD, Jennifer Doudna, PhD, and Cameron Diaz at the Breakthrough Prize award ceremony in Silicon Valley. Photo: Innovative Genomics Institute.
However, I-SceI can only target a specific 18-base-pair sequence that is not normally found in the human genome. Starting in the mid 1990s, researchers began engineering a new, customizable class of enzymes (called zinc-finger nucleases, or ZFNs) that could cut DNA like I-SceI, but would also be able to target any gene in any organism. ZFNs marked the beginning of an era during which we could, in theory, edit any gene in any way with unprecedented precision.
The most well-known use of ZFNs is the modification of the human CCR5 gene, which when expressed coats the exterior of T cells in the immune system. During most HIV infections, the virus will use CCR5 as an entry point into the immune system, where it will cause disease. When edited by ZFNs, modified CCR5 can block a large percentage of the virus from access to T cells. In a clinical trial, HIV-positive patients with genetically modified T cells showed promising responses to the treatment.
Despite the prospect of curing diseases like HIV and the reliability of these enzymes in gene modification, ZFNs haven’t taken the biomedical world by storm. The limiting factors for this are quite simple: ZFN design and production are both difficult and expensive. Designing ZFNs from scratch is a monumental endeavor for any researcher. Initially, it cost $25,000 for a single ZFN targeting a single gene, and the market had only one big producer, Sangamo Biosciences (full disclosure: I once worked for them). Before a substantial decline in the cost of ZFNs, a new method for gene editing took the throne.
The breakthrough
Unlike ZFNs, the CRISPR-Cas9 system is dead simple to design and orders of magnitude less expensive. In its natural setting, the CRISPR-Cas9 system is a bacterial defense mechanism against viruses. A bacterium can encode in its own genome small bits of viral DNA sequence, like a small database of known criminals. When a virus invades the bacterium, the CRISPR-Cas9 system will check to see if the invading viral sequence has a match in its database. If there is a match, the system will pair with and cut the viral DNA, thereby inactivating it.
In a 2012 publication in the journal Science, researchers from UC Berkeley and Umeå University in Sweden showed that they could reprogram the CRISPR-Cas9 system to pair with and cut a sequence of their choosing. Unlike ZFNs, which require algorithmic design followed by extensive validation, the CRISPR-Cas9 system only needs a unique sequence 20 bases in length in a gene where researchers want to generate a cut. Also, using CRISPR-Cas9 is no more expensive than normal laboratory costs.
Google Trends results for "CRISPR" (orange) and "ZFN" (blue) from 2004 to 2014. Higher y-axis value means more popular.
This discovery opened a floodgate for biological research, and the excitement is at a fever pitch. The scientists in charge of the original 2012 CRISPR-Cas9 study, Jennifer Doudna and Emmanuelle Charpentier, have been awarded the 2015 Breakthrough Prize in Life Sciences and have been discussed as possible Nobel recipients. Several startups have spawned and earned substantial venture capital from investors who see CRISPR-Cas9 as an industry worth billions in healthcare, biotechnology, and agriculture. And a heated patent battle has already begun over the ownership of the application of the technology.
The CRISPR-Cas9 system is now allowing researchers to study the genetics of a tremendous number of organisms at a rate never before seen. In the past two and a half years, publications using CRISPR-Cas9 have permeated scientific journals everywhere. It has been used in human cells, yeast, bacteria, mice, rats, hamsters, rabbits, goats, worms, parasites, mollusks, fruit flies, pigs, plants, fish, monkeys, salamanders, frogs, sea squirts, and silkworms.
In a 2013 publication, researchers showed the potential of this system for fixing genetic disorders. They studied adult mice with a mutation in the fumarylacetoacetate hydrolase (fah) gene, which causes an accumulation of toxic metabolites in the liver, resulting in severe liver damage. By injecting mice with the appropriate CRISPR-Cas9 components, the researchers were able to repair the defective fah gene and significantly reverse the effects of the initial mutation. In a separate 2014 study, researchers injected single-cell embryos of cynomolgus monkeys (also called crab-eating macaques) with CRISPR-Cas9 constructs targeting three separate genes at once. Ten babies were born, all of which had modifications at all three genes.
Number of publications in PubMed with the term "CRISPR" from 2002 to 2014. "ZFN" results could not be generated because they do not reach the 1,000 publication minimum threshold.
But there are most certainly caveats. One of the most significant problems with using any gene editing technology as a human gene therapy is the same as what makes it possible: cutting your genes. When the DNA of a mutant gene is cut, the DNA repair system can just as easily fix it as desired as it can make mistakes and make matters worse. ZFNs and CRISPR-Cas9 also have “off-target” effects, where rather hitting the right target, they cut at similar targets elsewhere in the genome, opening the possibility to mutate the genome further in unforeseen ways. These issues must be addressed if we are to edit our own genes (and such efforts are presently underway).
However, given that this technology is now only two and a half years old, its potential is immense. Gene editing does not stop at human health. Biotech companies can use it to produce genetically modified plants for enhanced crops. Synthetic microbes can be designed more easily to produce medicines or fuel. The largest potential impact, though, is likely in the basic biological sciences. Researchers have already warmly adopted CRISPR-Cas9 into their labs, and for good reason. It enables them to produce work previously too difficult, expensive, or unpopular. Its ease of use and low cost have effectively democratized genetics for biological researchers.
One of the first studies referencing CRISPR sequences in bacteria was published in the 1987, when the authors concluded their manuscript unsure of what they found: “the biological significance of these sequences is not known.”
It is now.
