This is the second article in an in-depth series on the impact of CRISPR/Cas9 genome editing technology on basic and clinical research, the biotechnology industry, and the world at large. GenomeWeb will be running the rest of the multi-part series over the next several months. The first installment, on the development of the CRISPR toolkit, can be found here.
NEW YORK (GenomeWeb) – Like many scientists, Arnaud Martin remembers the exact moment he first heard about CRISPR. For him, it was in 2013, reading a paper in the journal Genetics about using the new genome editing technology in Drosophila melanogaster. Arnaud was just a graduate student at the University of California, Irvine, working in the field of evolutionary developmental biology, or, evo-devo, but he soon moved to a postdoc position in the lab of Nipam Patel, a professor at UC-Berkeley, who was using RNA interference in small crustaceans called Parhyale hawaiensis.
Being at Berkeley, also the home of CRISPR/Cas9 pioneer Jennifer Doudna, excitement about the technology was in the air. Soon, Patel had Martin using CRISPR to knock out genes in P. hawaiensis. The results were immediate.
With RNAi, "It was a lot of work to knock down just one gene," Martin told GenomeWeb. "When we were able to use CRISPR, it was just mind blowing. We were able to knock out six genes in a few months."
Patel said that getting all six knockouts at a time was unthinkable just a few years ago. "You'd be doing it one or two genes at a time," Patel said. "We can interpret [a gene family] way more fully knowing the function of all six. We can think about how they interact and set up a pattern."
Patel's lab looks at correlations between gene expression and phenotype to answer questions both about developmental biology and evolution. P. hawaiensis is their organism of choice because it allows them to look at how evolution has developed different ways for animals to lay out their body plan. The wild-type crustacean has lots of legs, including two pairs of forward facing legs on the thorax for walking forward, followed by three very large pairs of thoracic legs that are curved backwards and used to scramble in reverse or spring backwards in an emergency.
"What we wanted to know is, 'What is the molecular genetic basis for defining all the leg types?'" Patel said. Previous studies of gene expression had indicated that a gene family, known as the Hox genes, was differentially expressed in the different body parts. Using CRISPR/Cas9, they were able to lay out how the Hox genes interacted and contribute to limb patterning. In one example, by knocking out expression of a single gene, they were able to make the big, forward curving legs into backward curving ones. "It not only gave us a phenotype, it gives us a mechanistic explanation," Patel said. "It gives us an amazing picture of how to generate diversity between species."
Martin and Patel published that study earlier this month in Current Biology. The paper is one of many examples of the far-reaching effects CRISPR/Cas9 genome editing is having in basic biological research, due to its effectiveness, efficiency, range, and straightforwardness.
"In the field of evo-devo, or generally biology that is about non-traditional organisms, CRISPR really has flavors of a revolution," Martin said. "It's really a game changer, completely opening the door to different sets of questions. It's creating so many possibilities it's hard to comprehend what people will do with it. I'm absolutely convinced it's going to take over the field pretty soon."
Among the benefits for evo-devo research that Martin and Patel brought up: it allows experiments in generation zero individuals, it allows complete gene knockouts versus partial knockdowns, it enables green fluorescent protein tagging just about anywhere, it allows insertion of DNA in addition to knockouts — potentially from related species with different evolutionary traits.
"You could propose an experiment now that before would have been a ridiculous proposal," Patel said. "Now you can do it because you technically know how to."
Beyond evo-devo
It's not just evo-devo that's going to feel the CRISPR effect. "It is like PCR," Jacob Corn, a professor at UC-Berkeley and managing and scientific director of the Innovative Genomics Institute, a joint venture between his school, Stanford University, and UC-San Francisco that promotes CRISPR. CRISPR, he predicts, is going to be a fundamental technique in molecular biology. "You never ask what are fields are affected by PCR, because it's all of them. In some ways, that's what we're talking about for genome editing," he said.
Scientists in fields such as evolutionary biology, stem cells, human molecular biology, neuroscience, ecology, and more will be able to answer questions they haven't yet thought to ask. And even those scientists working with canonical model organisms are being forced to re-think how they work in the age of CRISPR, according to Corn.
Whether it was obvious that CRISPR/Cas9 would work in just about every type of organism is a contentious question. UC-Berkeley and the Broad Institute are currently in a patent spat over CRISPR, and the Broad's success in that legal dispute may depend on making the case it wasn't obvious that a nuclease from bacteria would work in mammalian cells.
But, it's safe to say that CRISPR has worked just about everywhere it's been tried. In myriad organisms — not just Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Homo Sapiens, but also sea urchins, frogs, malaria parasites — and a multitude of cell types, CRISPR works.
The technology is so powerful that it has even helped scientists studying aging develop up a brand new model organism, the Turquoise killifish, which has a short lifespan but still shows the hallmark signs of human aging such as telomere erosion and cellular senescence.
"Any species in which you can collect eggs, and inject them, and have them survive, you'll probably able to get CRISPR/Cas9 to work," Patel said.
Because the Cas9 protein can be delivered with the programmable guide RNA (gRNA), a scientist doesn't even need to know anything about the molecular biology of the organism of interest, opening up the possibility to do experiments in just about every type of cell.
That's especially useful for evo-devo. Scientists interested in the morphology of animals are often drawn to insects, since they make up a staggering percentage of the number of species on earth. Insects demonstrate a tremendous diversity of shapes and adaptations, Martin said, but up until now there were just a few insects you could use to test genetic functions. "To manipulate genotypes, to modify your gene, to remove a gene or overexpress it — with CRISPR you can likely do it in any insect you're interested in."
Patel said interest in CRISPR was evident at a recent conference he helped organize, the inaugural meeting of the Pan-American Society for Evolutionary Developmental Biology, held in August at UC-Berkeley. He invited Corn to hold a breakout session on the use of CRISPR/Cas9, which was a big hit among attendees.
Corn, who called himself a cell biologist, said he has done lots of these kinds of sessions for many different subfields of biology and biomedical science. Toxicology, structural biology, systems biology, and DNA repair were just some of the fields he suggested could find ways to use CRISPR.
The impact of CRISPR has brought a huge initial shock, such as the seeming immediate ability to edit the human germline, but there's also a long-term, more diffuse aspect. "The long-term explosion of it is going to be all the unexpected stuff we suddenly learn about systems that were intractable before," Corn said.
"There are all kinds of caveats we've had to live with in modern cell biology," he said. "What genome editing is about is not having to make those compromises and instead ask molecular questions in the endogenous context, the cellular context.
When asked if there were any potential downsides to the surge in CRISPR, Corn did say that he's heard of cases where peer-reviewers asked to see gene knockouts with CRISPR when they wouldn't actually add anything to the experiment. "Usually they come to me in frustration and ask if we can help knockout the gene," he said. "In some ways it's like any fad. It's of course longer lasting than a fad.
"When RNAi came around everyone had to do RNAi," Corn said. "But then it disappeared from titles [and] it became just 'this is what you do.' There are going to be some years in which people are trumpeting, 'We're knocking out genes with CRISPR,' then eventually it's just going to become part of the toolbox."
But he also sees an upside to using CRISPR to make lots of knock outs. "When you do those studies, you're generating cell lines and those get banked," he said. The impending swell of cell lines has caused Corn and others to start thinking about whether they should bank them at all and instead just bank knowledge of the reagents used to create them. That means a potentially radical rethinking of how scientists use traditional model organisms, such as mice.
With CRISPR, it now only takes five weeks to create a knockout line of mice, Corn said. "That's almost no time," he said. "Some suggestions are that instead of banking gametes or embryos so we can bring mouse lines back, we should instead be banking knowledge of gRNAs [used to create them]."
"It's not totally clear what the ramifications would be," he said. "Which is more reproducible? To bank cell lines we use in the experiment or bank the reagents used to generate the thing?"
Meanwhile, scientists like Martin and Patel are answering questions that troubled Charles Darwin himself. Darwin had characterized the forces that governed what traits persisted in animals, but had no idea where new traits came from in the first place.
What's the difference between P. hawaiensis, an arthropod, which has the forward-curving pair of legs, and a roly-poly, an isopod, where all the legs curve backward? CRISPR/Cas9 can help answer that question, Patel said.
"One hypothesis is that regulatory elements drive evolutionary change," he said. "Now with bioinformatics, we can swap DNA between crustacean species. We could take a regulatory gene from an isopod and replace [the amphipod's version of the gene] with the isopod's. If limb morphology changes, we can prove it's the regulatory element" that was responsible for the change.
"CRISPR is making experiments like that much more within reach," he said.