Crispr and editing the human genome

Four years after its discovery, scientists worldwide are honing Crispr for a torrent of applications, pushing it from basic science to the field.

On February 1, 2016, a British researcher got the go from government authorities to genetically modify a human embryo using the gene editing technology Crispr-cas9. Just ten months earlier, Chinese scientists ignited a worldwide debate when they published their results of altering DNA in human embryos for the first time with the same technology. Is it legal to edit the human mark-up? Is it moral? Both studies, and the short amount of time elapsed between them, show the urgency of answering these questions, and the momentum and power the technology behind them has gained.

Crispr-cas9 is a force to be reckoned with. Four years after its discovery, scientists worldwide are honing Crispr for a torrent of applications, pushing it from basic science to the field. Researchers use it to edit the DNA of plants, animals and humans like a piece of text. It's being used by Harvard geneticist George Church to create an endless supply of organs for human transplant; MIT Professor Rudolf Jaenisch to create a fast supply of mice to study and fight dementia; and South Korean research fellow Jae-Young Yun to create disease-free food for the masses. This research may change who we are as individuals and as a species, and it's prompting both excitement and caution in the scientific world and beyond.

Crispr is an acronym for "clustered regularly interspaced short palindromic repeats." The technology was born about a decade ago when scientists unlocked the secret to bacteria's defence system. Bacteria produce two types of short ribonucleic acid (RNA) when they detect the presence of an invading virus. One of these linear-shaped RNA molecules is known as a guide, and is armed with a copy of the virus’s genetic code. It hunts down the invader, connects with the viral genome, and ushers in a special protein called cas9 when it finds its mark. From this point on it’s game over for the virus: cas9 cuts the viral DNA like a pair of molecular scissors and neutralizes it.

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Researchers Jennifer Doudna and Emmanuelle Charpentier were the first to publicly describe Crispr-cas9. In 2012, the pair harnessed the technology to edit purified DNA and their highly cited paper effectively kicked off the Crispr revolution. But it wasn't until six months later that the full potency of the gene-editing technology was realized.

In early 2013, George Church used Crispr to edit genes in human stem cells. He reprogrammed a squadron of guide RNAs to cut a DNA sequence at a precisely chosen location. The key to his discovery was integrating Crispr with a natural DNA repair mechanism called homologous recombination. This mechanism is necessary when both strands of DNA break: it mends itself by pairing with similar or identical nucleotide sequences. Using this premise, Church showed how Crispr could direct a nuclease to a specific section of DNA, and make and repair double strand breaks.

Church's study, and that of molecular biologist Feng Zhang from Broad Institute and MIT, was a major source of excitement for scientists worldwide. Zhang published a similar Crispr breakthrough at the same time as Church, showing how DNA can be unzipped, edited, and then zipped back up by the cell's repair team. Zhang’s lab notes show an earlier invention date than Doudna and Charpentier, winning him Crispr’s first patent in April 2014 – a decision now hotly debated and weighing on Crispr’s future commercial potential. But it didn’t impact research in the lab. A few months after Church and Zhang published their studies, Rudolf Jaenisch showed just how fast and accurate Crispr really was.

"A game-changer"


Jaenisch shaved years off the creation of transgenic mice. These are genetically engineered mice bred without one specific or many genes; they’re often used in disease research to study how each gene affects physiology and development. Jaenisch was the first to create a genetically modified mouse in 1974, and in May 2013, he was also the first to create a one-step generation of mice with multiple edits: “Crispr-cas9 enables us to do genome editing in a way that was much more difficult before. It's a major change for medicine,” he says. The mouse strain took him three weeks instead of two years to create, making it an extraordinary feat. The conventional way of studying genes before Crispr was agonizingly slow: Scientists would insert DNA into a mouse’s embryonic stem cells and breed them. It was both a cumbersome and limited approach, and would often only reveal one characteristic, in one gene, in one generation of mice. Hundreds of genes work together in illnesses like cancer, autism, and Alzheimer’s, so it’s important to not only understand the basic functions of a gene, but also how they interact. Crispr – with its ability to edit multiple genes within a matter of weeks – proved itself a huge advancement for studying complex diseases. Jaenisch says: “It really was a game-changer.”

Since these first few ground-breaking studies, Crispr's potential has become more evident by the day. The number of research papers using the technology tripled in the last two years. Patent applications for Crispr exploded in 2014, and in late 2015, $120 million in funding was poured into Editas Medicine alone. Editas is one of the many emerging Crispr-focused biotech startups, and recently announced they intend to start human trials within two years. Their first clinical trial will aim to treat a rare genetic retinal disease that causes blindness.

Pig organs for human transplant


Crispr breakthroughs are now appearing by the month. In October 2015, George Church took great strides in rendering pig organs fit for human transplant. More than 120,000 people in the United States alone are waiting for a life-saving organ transplant, but the infectious porcine endogenous virus has stymied researchers in this field. Pigs harbor this virus in multiple sections of their DNA, and it can infect the kidneys of transplant patients upon contact. But Church says he and his team eliminated the threat in one fell swoop: “We used CRISPR to cut and inactivate all 62 viruses permanently. Prior to this it was considered very challenging to edit merely 3 genes at once.”

One month later, Crispr helped to end a 30-year quest: researchers wielded the technology to engineer mutant malaria-free mosquitoes. An international team edited mosquitoes' genes to resist the malaria-causing parasite, and pass it on the trait to the majority of their offspring.

December's big breakthrough saw three teams of scientists get closer to treating a rare and fatal disease. Hundreds of boys are born with Duchenne muscular dystrophy every year, which is a genetic disease that leads to weakness, atrophy, and eventually, a young death. In three separate studies, the scientists successfully used of Crispr to treat the disorder in mouse models.

While the prospective outlook for Crispr is promising, the speed at which the technology is advancing has prompted caution from scientists, doctors and bioethicists. Crispr is still young and raises a number of unanswered questions: Editing genes could cause unintended mutations, or introduce changes elsewhere in the genome. Some researchers are also concerned Crispr could be used to create designer babies, whose genetic make-up is defined by desirable traits. For this reason, the clinical application of Crispr in humans has caused a stir. In April 2015, Chinese scientists reported genetically modifying the genomes of human embryos. They targeted a gene responsible for β-thalassaemia, a potentially fatal blood disorder, and claimed the embryos they used could not have resulted in a live birth. The experiment failed, but it sparked a debate regardless. Researchers gathered in Washington D.C. in December that year to discuss the science and ethics that Crispr entails. In February, the British Human Fertilisation and Embryology Authority (HFEA) approved Kathy Niakan’s license application to study human embryos donated by couples with surplus embryos after IVF treatment. The stem cell scientist at the Francis Crick Institute in London will study which genes are at play in the early days of human development by selectively switching them on and off.

Jaenisch believes that the basic science behind gene editing in embryos should not be inhibited. Its application in humans, however, is a different story. “This technology not only raises scientific questions but poses serious ethical issues: do we want to edit the human genome? It’s very complicated and the many different parts of society should have input in this,” he says. George Church points out that an FDA approval system is already set up to help facilitate decisions on this. He says: “We need to be moderately cautious about both gametes and adult somatic tissues – basically like all new medical devices and therapeutics.”

Saving the banana


Just as gene-editing has the potential to change an entire population, it could also alter an ecosystem with unpredictable results. Yet if done with caution, Crispr paves a promising path for food security. Last October, a team from Seoul National University created disease-resistant lettuce while skirting genetically modified organism (GMO) regulations. GMO regulations are in place to avoid foreign DNA left in the plant, which can enter the food chain and potentially change entire ecosystems. But foreign DNA wasn't a part of this team's Crispr toolbox: “Our CRISPR-Cas9 RNP strategy circumvents all of those concerns,” says plant geneticist Jae-Young Yun from the Institute for Basic Science in South Korea, who was involved in the study.

Yun envisages a future where many breeders will utilize their work to create food that’s not susceptible to disease. If successful, it holds the promise to end food shortage and tackle world hunger. But until then Yun has his sights on another problem: saving the Cavendish banana. Global plantations of this household banana are currently battling a lethal fungus called Fusarium wilt. It's looking likely to wipe out the banana as we know it, as a similar fungus wiped out its predecessor, the Gros Michel banana, some 50 years ago. Yun and his team want to find the genes susceptible to fungal infection, and remove them from the banana's DNA using Crispr.  Yun says: “We plan to make Fusarium-resistant Cavendish bananas before this cultivar becomes extinct.”

While the US National Institutes of Health don’t fund gene editing research with human embryos at present, other experts have a similarly positive outlook as Yun. When asked what Crispr advancements Church is looking forward to in the coming years, he responds: “I hope to see several examples of anti-viral therapies, including in hepatitis, herpes, and HIV.” Rudolf Jaenisch thinks along the same lines, and adds work in this area is well on its way: “Crispr is already being used in bone marrow cells to help knockout a person’s AIDS receptor. This means it could essentially help cure people of the disease.”

 

Timeline of Crispr studies:


2012


June




2013


January




May




December




2014


January




July




October




2015


March




April




October




  • Crispr is used to inactivate PERVs in pig genes, advancing the use of pig organs in human transplant


November




December




Image courtesy of Stefano