What is it being used for?
In the past few years, there has been talk among the scientific community about CRISPR and the many possible benefits it might present. However, not all the talk has been positive. There are some concerns about the dangers it poses. What is CRISPR? What can it help us with? What should we be worried about?
Every cell in our body contains DNA, which has many millions of base-pairs, or nucleotides. The way these are fitted together forms a language that tells our bodies to function in specific ways. Some of these nucleotides are codes for genes. There are 40,000 proteins that become outputs of those genes and they’re involved in our health, wellbeing, and any defects that can be problematic or cause disease. Even though we’ve had a detailed map of the human genome since 2003, we don’t know the complete function for each gene. For years, scientists have been looking for ways to understand this and to find ways to correct any imperfections by altering the affected genes without adversely impacting the DNA. This concept is known as gene editing. Some of the early methods were viral gene editing, gene replacement, and Transcription Activator-Like Effector Nuclease (TALENS). While effective, it was time-consuming and costly. Also, it was only able to edit large chunks of genetic information rather than specific sections. A newer method, CRISPR-Cas9, is more precise, easier to use, and four times more efficient than TALENS. This meant that instead of cost thousands of dollars and taking weeks or months to alter a gene, it costs under $100 and only takes a few hours. One of the fascinating things about CRISPR is how quickly everything is developing. In 2011, there were fewer than 100 published papers on CRISPR. By 2018, there were more than 17,000. Since the initial descriptions and papers were written, this technology has exploded, resulting in refinements with new techniques being developed and improvements in precision.
The concept of CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeat) was discovered in 1987, but its significance to gene editing wasn’t realized until 2007. A CRISPR is a specialized stretch of DNA that has two distinct characteristics: the presence of nucleotide repeats and spacers. Repeated sequences of nucleotides are distributed throughout a CRISPR region. Spacers are bits of DNA that are interspersed among these repeated sequences. The interesting thing is that there are numerous CRISPRs in each organism. To edit genes via CRISPR, you need a protein that acts like a pair of molecular scissors capable of cutting strands of DNA. The most commonly known protein is Cas9, but other examples are Cpfl or Cas13, which can edit RNA (DNA’s sister). The process was first observed in bacteria. When a bacterium is attacked by a virus, it captures bits of DNA from the invader and uses them to create DNA segments known as CRISPR arrays. They allow the bacteria to “remember” the virus or closely related ones. So, if the virus attacks again, a portion of the CRISPR is transcribed and turned into CRISPR RNA, or “crRNA.” Each crRNA consists of a nucleotide repeat and a spacer portion. The protein (Cas) typically binds to two RNA molecules: crRNA and tracrRNA (or trans-activating crRNA). The two then guide the Cas to the target site where it will make its cut. This expanse of DNA is complementary to a 20-nucleotide stretch of the crRNA. Using two separate regions, or domains, on its structure, Cas cuts both strands of the DNA double helix, making a double-stranded break, which chops up and destroys the DNA of the foreign invader. To ensure that Cas doesn’t just cut anywhere in a genome, short DNA sequences known as PAMs (protospacer adjacent motifs) serve as tags and sit adjacent to the target DNA sequence. This means that if Cas doesn’t see a PAM next to its target DNA sequence, it won’t cut. The CRISPR-Cas9 system in other organisms works similarly. In 2012, two pivotal research papers were published and concluded that Cas9 could be directed to cut any region of DNA in any organism. All that was need to do this was to change the nucleotide sequence of crRNA. The system fuses crRNA and tracrRNA together to create a single “guide RNA.” So, gene editing requires only two components: a guide RNA and the Cas9 protein.
CRISPR technology is proving to be a simple yet powerful tool for editing genes. It’s likely to speed up genome screening and genetics research could advance immensely as a result. Using it, researchers have figured out what different genes in different organisms do. For instance, by removing individual genes, they’re able to see which traits are affected. Also, it has worked on every organism that it has been tried on, which means it could revolutionize everything from medicine to agriculture. The technology has been applied in the food industry to engineer probiotic cultures and to vaccinate industrial cultures against viruses. These uses aren’t surprising since the original discovery of CRISPR immunity came from researchers at Danisco, a company in the food production industry. Danisco scientists were studying a bacterium, Streptococcus thermophilus, which is used to make yogurts and cheeses. Some viruses can infect this bacterium and damage the quality or quantity of the food. It was discovered that CRISPR sequences equipped S. thermophilus with immunity against these viral attacks. Manufacturers can apply the same principles to improve culture sustainability and lifespan to other foods. In agriculture, the technology is being used in crops to improve yield, drought tolerance, and nutritional properties.
When it comes to humans, there are two main methods that scientists are looking at. The first is taking cells out of the body, manipulating them in the laboratory to either remove a defective gene or add/enhance an ability by turning on a gene or fixing it before putting those cells back in the body. The second area is injecting something into the body, which can edit people’s genes so that they can either be turned on or off. There are over 7,000 monogenetic diseases or diseases that can trace back to a single gene defect. Monogenetic diseases fall into two categories, toxic gain of function and toxic loss of function. Toxic loss of function means the gene has got a flaw in it; the person loses the function of that protein and the result is the disease, such as sickle-cell anemia. Toxic gain of function is the mutation causes extra genetic material to be present and this causes the disorder, like transthyretin. Other monogenetic diseases would be cystic fibrosis, beta-thalassemia, glycogen storage disease, Behçet’s disease, and Fabry disease. The goal of CRISPR would be to modify the genes to treat the diseases. In 2013, the first reports of using CRISPR to edit human cells in an experimental setting were published. They confirmed that the technology could be useful in correcting genetic defects. The reason it works is that once the DNA is cut, the cell’s natural repair mechanisms kick in. There are two ways this happens. One process involves gluing the two cuts back together, which is known as non-homologous end-joining. Unfortunately, this way tends to introduce errors because nucleotides are accidentally inserted or deleted, resulting in mutations. In the second technique, the break is fixed by filling in the gap with a sequence of nucleotides. To do this, the cell uses a short strand of DNA as a template. With CRISPR, scientists can supply the DNA template of their choosing so that they can correct a mutation. The success of the technology was validated in December 2017 when the Food and Drug Administration (FDA) approved a gene therapy for the treatment for the first time in the nation’s history. The therapy was developed by researchers at the University of Pennsylvania and Children’s Hospital of Philadelphia to treat a rare, inherited form of retinal blindness. It’s known as Luxturna (voretigene neparvovec-ryzl) and significantly improves eyesight in patients with confirmed biallelic RPE65 mutation-associated retinal dystrophy. Individuals who have this mutation become completely blind by mid-life, so the approval of a treatment was a life-changing moment.
Besides genetic diseases, another area of research is oncology. Most people think that cancer is an incredibly hard disease to fight. While that is currently true, scientists are hoping to change that. They’ve known for a while that our immune systems have the ability to fight cancer cells and essentially dissolve micro-tumors. The problem is cancer is clever because it evades the body’s immune system by becoming invisible due to certain proteins that are created as checkpoints to interfere with the immune system attacking ourselves. This means they mimic our own cells by taking advantage of these checkpoints. With CRISPR, the thought is by removing immune cells from the body, applying the technology to turn off these checkpoints and putting those immune cells back in the body; then, those immune cells would attack the tumor.
Areas that have tremendous potential for using CRISPR are antibiotics and antivirals. Globally, we’re running low on effective antibiotics as bacteria evolve and are becoming resistant to them. This is happening due to the widespread use of prescription antibiotics and the use of antibiotics in animal food production. Microorganisms advance quickly and many have developed defenses against the medicines designed to kill them. Every year about 700,000 people around the world die from infections that don’t have an effective treatment. According to United Nations estimates, the number could rise to 10 million by 2050. The reason for the dramatic jump is that it’s difficult and costly to develop new antibiotics, so many pharmaceutical companies aren’t willing to invest in them. Viruses change quickly, as well. They find new ways of disguising themselves from drugs, often by hiding inside host cells. In addition, less than 100 antiviral drugs have successfully made it to consumer use since they first were approved in 1963. This is where CRISPR could come into play. One study showed that researchers could successfully use CRISPR-Cas9 to eliminate a species of Salmonella. They did this by programming CRISPR so the bacterium viewed itself as the enemy. This forced the strain of Salmonella to make lethal cuts to its own genome. The team began with a conjugated plasmid, which is a small packet of genetic material that can replicate itself and be passed from one bacterium to the next. To the plasmid, they added encoded instructions that targeted the Salmonella’s DNA. The “improved” plasmid was then placed inside E. coli bacteria because it’s typically part of a healthy gut microbiome, which would mean that it would already be present if a person ingested pathogenic Salmonella. If this happens, the E. coli could then transfer the engineered plasmid to the Salmonella, where the CRISPR instructions would activate, destroying the harmful bacteria. In the petri dish, this is exactly what the researchers observed. Using this method would allow scientists to harness the power of the human body’s resident, good microbes in preventing disease.
At the University of California San Diego, scientists have developed a new CRISPR-based gene-drive system. It radically boosts the efficiency of inactivating the gene that renders bacteria antibiotic-resistant by working with preferred traits called “active genetics.” The new “pro-active” genetic system, or Pro-AG, features a modification of the standard CRISPR-Cas9 gene-editing technology. The new system addresses the issue of antibiotic resistance presented in the form of plasmids. Multiple copies of (or amplified) plasmids carrying antibiotic-resistant genes can exist in each cell and can transfer antibiotic resistance between bacteria. Pro-AG uses a cut-and-insert repair mechanism to disrupt the activity of the antibiotic-resistant gene. The result is the insertion of tailored genetic material into target sites with high precision. In a study, the researchers were able to demonstrate the effectiveness of the new technique in experimental cultures containing an increased number of plasmids carrying genes known to have resistance to the antibiotic ampicillin. Pro-AG is not yet ready for treating patients, but the hope is that a human delivery system carrying Pro-AG could be deployed to address conditions and infections. Besides, if combined with a variety of existing delivery mechanisms, the technology could be useful in removing antibiotic-resistant strains of bacteria from the environment, such as sewers, fish ponds, and feedlots. Since Pro-AG “edits” its targets instead of destroying them, the system would enable scientists to manipulate bacteria for a broad range of future biotechnological and biomedical applications, which wouldn’t only leave them harmless, but might even transform them into performing beneficial functions.
Another CRISPR enzyme, Cas13, is being looked at for detecting and killing three different single-stranded RNA viruses that infect human cells. These are influenza A virus, lymphocytic choriomeningitis virus, and vesicular stomatitis virus. In the case of the flu virus, Cas13 reduced its infectiousness by over 300-fold. If researchers can do this for three relatively mild human viruses, they should be able to use the technology to treat more deadly viral infections as well. By developing such treatments, it could allow scientists to eradicate certain bacteria more precisely than ever. This would be incredibly valuable since conventional antibiotics do not distinguish between good and bad bacteria. Instead, they eliminate everything indiscriminately, which can create other problems, especially for those with weakened immune systems. Compared to current antibiotics and antivirals, CRISPR has the advantage of being easy to tweak as needed. For instance, if a virus evolves and mutates, the system can be changed to match whatever the microbe is doing. Figuring out delivery mechanisms is still a challenge that has yet to be worked out. CRISPR-based antibiotic pills aren’t anywhere near pharmacy shelves because researchers need to prove that they’re effective in living animals and humans. Also, they need to demonstrate that CRISPR-based treatments will be cheaper than conventional therapies.
However, it’s not all just positive news surrounding CRISPR. There are several ethical concerns about the consequences of tampering with genomes. Part of the worries arises from the fact that CRISPR isn’t a hundred percent efficient, which is why most scientists are urging caution on human testing until more is known about potential long-term impacts. CRISPR’s ability to inflict mayhem on DNA is dangerously underestimated. Occasionally, the Cas9 enzymes can edit DNA in unexpected places. This is known as “off-target effects” and can lead to unintended mutations, which might lead to cancer or even create new diseases. Some scientists point out that even when the system cuts in the right spot, there’s a chance of not getting a precise edit. Also, there’s scientific evidence that some people have naturally occurring immunity to CRISPR, which means that their bodies produce substances that actually will turn off any kind of CRISPR that’s put into them.
One of the biggest fears related to CRISPR is genetic drive. This means that manipulated genes get incorporated into the genome inside cells and those genes could be transferred to other organisms altering an entire species. In one sense, scientists are contemplating doing this to help control elements in the environment. Normally, when an organism mates, there’s a 50% chance that it will pass on any given gene to its offspring. By using CRISPR, scientists can alter these odds so that there’s almost a 100% chance that a particular gene gets passed on. With genetic drive, scientists could guarantee that an altered gene spreads throughout an entire population in short order. An example is genetically modifying Anopheles gambiae mosquitoes to make them resistant to malaria, which would prevent its transmission to humans. Another option is enhancing sterility among female mosquitoes, which would ultimately kill the entire species. This concept could also be used to eradicate invasive species and reverse pesticide and herbicide resistance. The problem is that the altered trait could spread beyond the target population to other organisms through crossbreeding. The method could reduce the genetic diversity of the target population as well. In either case, entire species could be wiped out and ecosystems overturned. When it comes to humans, genetic drive means the manipulations get passed generation to generation to generation. Due to the possible off-target effects, this means that unintended mutations resulting in diseases that might not be known about until after it’s already widespread, making them very difficult to control or get rid of.
Another major concern with CRISPR is germline editing. This is making genetic modifications to human embryos and reproductive cells, like sperm and eggs. While it might seem like a good idea to eliminate diseases, there could be some potential problems with permanently altering the human genome. Part of the problem is these genetic changes could be difficult to undo and raises the ethical question of whether or not certain things need to be “fixed” or if we should be making changes that will impact future generations without their consent. A considerable component of editing the human genome is the possibility to create “designer babies.” This means using CRISPR technology to enhance standard human traits in the hopes of improving athleticism or gaining superior intelligence. Some people feel that the ability to make “ideal” human beings could open the door to eugenics, which is the removal of “undesirable” genes from a given population. The practice of eugenics was widespread in the early to mid-20th century. This is generally viewed as a dark spot on the history of medicine. Based on these apprehensions, germline editing is currently illegal in many countries. The US government will not fund any genomic editing of human embryos. However, it’s not unlawful for researchers in the US to secure their own funding to move forward with their experiments.
In 2017, the US National Academies of Sciences, Engineering, and Medicine held an international summit of scientists, ethicists, and others to discuss germline editing. They issued a comprehensive report with guidelines and recommendations regarding it. They urged caution in pursuing germline editing, but emphasized that “caution does not mean prohibition.” Their advice is that germline editing should only be done on genes that lead to severe diseases and only when there are no other reasonable treatment alternatives. Some criteria that they insist should be kept track of is data on the health risks and benefits. The group also maintains the need for continuous oversight during clinical trials and following up on families for multiple generations. Some scientists have even called for a cessation of germline editing research until more is known. In addition, they want better regulations. For example, they point to the process of drug development around the world. In the US, the FDA closely monitors the safety of any investigational drug. Other countries follow similar procedures. The scientists feel that only after “much more research to meet appropriate risk/benefit standards” with “broad participation and input by the public” should regulations be issued and further steps toward germline editing are taken.
However, not everyone feels this way. China doesn’t have the regulatory and ethical safeguards that other countries do, which means their researchers have begun working on human embryos and have proceeded to human clinical trials using CRISPR. Typically, clinical trials are broken into set stages that allow a new drug or therapy to be closely observed for risks and benefits. After identifying the potential in the lab, the first stage involves testing the drug/therapy in animals to make sure that there’s complete safety. Next, it goes into very limited testing in human beings before being more broadly tested in humans. If the product passes all of these steps (which can take several years), it’s released for widespread use. If it doesn’t pass, it can’t be used. The problem with CRISPR technology in China is they took the animal data and went right into widespread trials in human beings. One area in which they’ve done this is cancer therapy. As of right now, it’s too early to tell if it’s successful or not.
There’s no question that many scientists feel experimentation of CRISPR technology in humans is premature. This is why in November 2018, the world was shocked that a Chinese scientist, Dr. He Jiankui, reported that he had created the world’s first human babies with CRISPR-edited genes. Dr. Jiankui said that he made a set of twin girls who are resistant to HIV. Dr. Jiankui described his experiment at an international gene-editing summit in Hong Kong. During the announcement, he also revealed that another early pregnancy with a genetically-modified fetus is underway. According to the data, he focused on a gene, CCR5, which HIV uses as a way to infiltrate human cells. To prevent this from happening, several scientists have tried removing the immune cells of HIV patients and deactivating CCR5 using gene-editing techniques before injecting the cells back into the body. In Dr. Jiankui’s experiment, the twins’ father is HIV-positive, so Dr. Jiankui and his team decided to deactivate the gene before the embryo was implanted into the mother via in vitro fertilization (IVF). Some scientists say his rationale for doing this was extreme and unfounded. They say this is especially true since the likelihood of either twin being HIV positive was rare and neither one was shown to have HIV before the gene was edited. Critics of the experiment also point out that there are medications that can block CCR5 and have been proven to be safe and effective after rigorous clinical trials. Also, the transmission of the virus can be reduced significantly through safe-sex education. Also, it’s important to note that CCR5 isn’t the only way HIV can enter the cells since different strains can use other genes. Basically, Dr. Jiankui tried an untested therapy on a normal gene and provided no benefit to the twins. Furthermore, he might be setting the twins up for future health problems because people who have a natural deficiency in CCR5 appear healthy, but there is some evidence that they might be more susceptible to West Nile virus and are more likely to die if they catch influenza.
Another problem scientists have with Dr. Jiankui’s work is that it’s sloppy. For example, it appears that he only managed to edit half of one of the twin’s CCR5 genes, with the rest being normal. This could happen for two reasons. The first is that every cell in her body has one standard copy of CCR5 and one edited one, which means she’s heterozygous. The other is that half of her cells carry two edited genes and half carry two normal ones, which means she’s mosaic. If it’s the first, she wouldn’t be resistant to HIV. If it’s the second, it depends on whether her immune cells specifically carry the edits. The same might apply to the other twin because, based on the data, she seems to have regular copies of CCR5 somewhere as well. In addition, Dr. Jiankui made new mutations to the twins’ genomes that are substantial changes and could alter how CCR5 works.
The methods Dr. Jiankui and his team used to enroll participants in the study have also been called into question. They relied on an AIDS association to reach out to patients by telling them that study was an “AIDS-vaccine development project.” When it came to the informed-consent process, Dr. Jiankui said he and another professor personally took the participants through the form. Getting consent is a specific skill that requires training neither one of them had. Also, the consent document uses very technical language and appears to be similar to a form a business would use when subcontracting. It also gives the team the rights to use photos of the babies in magazines, calendars, billboards, product packaging, and propaganda. One of the participants who dropped out of the experiment says that he wasn’t informed about the risks regarding off-target effects or that gene editing was a prohibited and ethically controversial technology.
Besides not informing the participants of his true intentions, Dr. Jiankui also did not tell his employer, the Southern University of Science and Technology. In fact, he took unpaid leave to work on the project. As a result, the university plans to launch an investigation into the venture, which it called a “serious violation of academic ethics and standards.” Jiankui states that he received approval from Shenzhen Harmonicare Hospital to conduct the experiment. Officials from the Medical Ethics Committee at the hospital indicated that they never met to discuss the endeavor and that the signatures on the approval form “are suspected to have been forged.” Most likely, it was the financial incentive from the two biotech companies that he’s affiliated with that drove Jiankui forward with the experiment.
The reaction from the scientific community to Jiankui’s announcement was swift and negative, with many scientists feeling that the experiment was deeply disturbing and ignored ethical norms. Some wondered if Dr. Jiankui was telling the truth? If he was, does this mean that a hold needs to be placed on CRISPR research until better global regulations are created? This might be the case since a different group of scientists revealed that they revived a virus called horsepox, which other researchers stated would make it easier to recreate far more dangerous viruses, like smallpox. Both of these examples prove the real vulnerability of CRISPR is that without regulation, small groups of researchers can make decisions about doing experiments that have potentially global consequences and everyone else only learns about it after the fact.
All of the hurdles related to CRISPR need to be addressed before it can be used on a wide-scale. The first is the lack of knowledge about the potential harms that could result from editing human genes not only in the short-term but the long-term as well, such as if it can result in an amplified chance of developing cancer. The second issue is ensuring the technology doesn’t mistakenly attack genes other than the one’s being targeted. The third item is the implementation of regulations and guidelines to prevent the technology from moving to human testing before it’s fully vetted and understood. This means regulatory framework should address safety mechanisms, risk of multigenerational side-effects, ethical hurdles regarding human embryos, and any equity concerns. Without sufficiently addressing these matters, the technology has the potential to be incredibly dangerous. To protect all of us, scientists, ethicists, politicians, and other officials need to come together and devise a way forward that allows the benefits of the CRISPR to be fully realized while minimizing the risks.