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Genetic Linkage

A Conversation with CRISPR-Cas9 Inventors Charpentier and Doudna

CRISPR-Cas9 works like scissors on double-strand DNA. (NHGRI)
At the American Society of Human Genetics meeting in October, CRISPR-Cas9 inventors Jennifer Doudna and Emmanuelle Charpentier accepted the Gruber Genetics Prize, then stopped by the press room. For me, this was a little like sitting down with Bono and Bruce Springsteen, but the women were wonderfully down-to-earth, and a little stunned at all the attention since they published their key paper in 2012 on the technique that is speeding gene editing and making genome editing a reality.

This week an International Summit on Human Gene Editing held in Washington DC discussed the potential promises and pitfalls of gene editing technology. A terrific review is here. For those of us who were around at the debut of modern biotechnology in the 1970s, it’s déjà vu all over again. I hope the outcome will be the same. Although concern over recombinant DNA technology back then began with alarm, it basically ended with not triple-headed purple monsters, as my then-grad-school advisor dubbed the concern, but with a new and more targeted source of drugs, beginning with human insulin.

Below are selected comments from Drs. Doudna (a Howard Hughes Medical Institute Investigator and professor of molecular and cell biology and chemistry at the University of California, Berkeley) and Charpentier (director of the new Max Planck Institute of Infection Biology in Berlin) from their talks and visit to the press room in October. I’ll cover here what I didn’t a few weeks ago here and in Medscape to accompany the conference.

RL: Why is CRISPR-Cas9 taking off right now?

Jennifer Doudna (JD): I watched a video by Bill Gates and Steve Wozniac from the beginning of the personal computer age 25 years ago. When they were asked when they realized the PC was going to take off, they said it was serendipitous, because society was at a point where people were ready and eager to adopt that technology. That’s a very interesting parallel to CRISPR-Cas9.

We had the first bacterial genome in 1995, that’s 20 years ago. And with all the genome-wide association studies and human genome sequencing since 2000, we’ve built up an appreciation for the kinds of mutations that cause disease and the desire to be able to manipulate genes beyond systems like yeast and worms. We’re seeing the convergence of those technologies with an efficient and easy way to manipulate genes.

If this had happened 10 years ago we might have seen a different trajectory. In pubmed (for the 2012 paper) it’s exponential: 120 citations the first year, 400 in 2013, 600 in 2014, and more than 1200 as of October 2015. There was a pent-up need for the technology to manipulate genomes to be easy, and that’s what we’re seeing now.

RL: How did your research paths converge?

Emmanuelle Charpentier (EC): I was trying to understand how bacteria cause infectious diseases, from the pathogen and the human sides, particularly Streptococcus pyogenes. It causes necrotizing fascilitis, toxic shock syndrome, myositis, tonsillitis, pharyngitis, impetigo, cellulitis, scarlet fever, rheumatic fever, reactive arthritis, and rheumatic fever. I was also interested in infection of bacteria by invading genomes. Mobile genetic elements (bacteriophage) attack a bacterial host, and the host has a defense against the invaders that is considered the innate immune system of bacteria.

JD: Precision-editing a genome isn’t a new idea, it’s been around for decades. In the 1980s, as a grad student, I was working on double strand DNA break repair. The field of genetics has long appreciated that the ability to make changes in DNA would be an incredibly useful tool. The 2007 Nobel Prize in Physiology or Medicine (to Mario Capecchi, Martin Evans, and Oliver Smithies) was for harnessing homologous recombination, one of two DNA repair pathways activated by double strand breaks, to create the knockout mice that have since served as models for many human genetic diseases.

Our own journey was going from the basic science from Emmanuelle through our realization that we had come across a pathway in bacteria that could be harnessed as a powerful genome engineering technique. I started with a small NSF grant 10 years ago, before anyone thought this would be useful for anything.

EC: I started to work on this in 2006, using bioinformatics, then more seriously in 2009, with this paper in 2011.

RL: How Does CRISPR-Cas9 Work? The short version, that is.

EC: The enzyme Cas9, an endonuclease, is programmed with a guide RNA to target and cleave a specific DNA sequence at two strands. The manipulator just needs to engineer the guide RNA according to the sequence of the gene to be modified.

JD: Bacteria defend against viral infection by acquiring little bits of DNA from viruses into their genomes, making RNA copies of viral sequences, and incorporating them into one or more proteins used to target the viral DNA. Then the RNA-protein complex finds double-stranded regions, unwinds them, and positions itself so two active sites can cut the double-stranded DNA at a precise, targeted sequence. Cells recognize double strand breaks and repair them using two pathways that add new sequence or heal the old. It is a remarkable molecular machine that can search through large slots of DNA to find a particular sequence.

EC: The idea was relatively simple: genome editing with sequence-specific nucleases inducing a double strand DNA break at a specific site. The RNA-programmable CRISPR-Cas9 allows precise surgery in the cells of many organisms, including mice, plants, monkeys, and humans.

JD: Bacteria use CRISPR-Cas9 to cut a viral DNA sequence, but scientists harness it to make double strand breaks where we might like to introduce a small change in the genome.

(Dr. Doudna explains the nuances of CRISPR-Cas9 in this video.)

RL: The ease of deploying CRISPR-Cas9 raises concerns that it'll be used to alter the genome of a fertilized ovum. In April, researchers from Sun Yat-sen University published that they’ve already done this. Why the concern over germline modification?

JD: When I saw the publication in early 2014 of germline editing in monkeys, it came home to me that there’s no reason to think it couldn’t also be used in humans. Why not? That raises ethical questions as well as considerations about the utility for applications where it’s easy to employ, yet we as scientists should take a step back and say “should be go there?” Those thoughts are what launched me on the path I’m currently on in bringing colleagues on board to discuss the bioethics openly.

Doing somatic (body) cell editing in adults has inherently more immediate applications because we don’t have to think about the ethics of passing on heritable mutations. On the other hand, in some ways it will be harder to do because we have to deliver to adult tissues. Ironically, germline application is a lot easier to deliver. If we know there is an inborn genetic error, it could be more efficient and safer to correct it at an early stage of embryonic development than if we wait to do it in an adult patient.

RL: What are some non-medical uses of gene editing?

JD: Gene drive technology is an approach using CRISPR-Cas9 that could lead to elimination of species by changing organisms in ways that make them sterile, such as mosquitoes. It’s not science fiction anymore, it’s here right now. (A recent paper details using CRISPR-Cas9 to create malaria-resistant Aedes aegypti mosquitoes)

A researcher is using CRISPR-Cas9 to study the genetic changes in going from a mouse to a jerboa, a hopping desert rodent. It has huge hind legs and is bipedal. A jerboa is genetically very similar to a mouse, but clearly different in phenotype. Until using CRISPR to interrogate the genome of this organism, it was completely intractable genetically. We can now introduce changes to that organism and possibly reconstruct evolution.

Third-graders are using CRISPR-Cas9 to change a DNA sequence in yeast. A colorometric assay manipulates yeast that turn blue. It is a genome engineering change that is so simple to use. For $65 you can order reagents from AddGene, the “nonprofit plasmid repository,” and in a week or two have genome-modified cells. It’s phenomenal. (I was unable to locate a kit targeted especially for students although I suspect the folks at Carolina Biological Supply are onto it.)

EC: There’s an interesting debate about using CRISPR-Cas9 on plants to create GMOs. That’s very restrictive in Europe. There they may not accept CRISPR-Cas9 in any plant, because they may not consider plants that have deleted genes to be non-GMOs. Decisions will be made in Europe by the end of the year.

JD: The US Department of Agriculture ruled that if a genetic manipulation results in a knockout, it’s not a GMO.

HISTORICAL ASIDE FROM RL: Those of a certain age will recall the famous Frostban “ice-minus” bacteria, common Pseudomonas with a gene removed that, when put on strawberry plants, enables them to survive frost. Environmentalists successfully blocked the first field tests, although the manipulated bacteria merely mimicked a naturally-occurring mutation. An article in The Scientist chronicled the hysteria with some priceless anti-science quotes. My favorite: a plea that the genetically deficient bacteria would cause “"more death and destruction than all the wars we have ever fought." But of course designing babies is different from helping strawberries.

JD: These concerns mean there’s a real need to discuss the science. I met a researcher who’s working in South Korea on bananas, and I’m from Hawaii and I know banana farmers. Bananas are genetically very similar to each other and that makes them highly susceptible to disease. Currently a fungus is wiping out bananas. In the end it may come down to a choice, not whether we want GMO or non-GMO bananas, but whether we want bananas or not? If we want bananas, people might have to accept a fungal resistance gene that has been introduced.

All of these areas are exciting and require serious consideration before forging ahead.

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