She Has Her Mother's Laugh: The Powers, Perversions, and Potential of Heredity (65 page)

“
We had built the means to rewrite the code of life,” Doudna later recalled.

After Doudna and her colleagues published the details of the experiment in 2012, a CRISPR scramble began. Her team, as well as others, tried to get the CRISPR molecules into living cells. Researchers learned not only how to cut out pieces of DNA from those cells but how to repair it as well.

In one of these experiments, Feng Zhang and his colleagues at the Broad Institute in Cambridge, Massachusetts, delivered a pair of CRISPR systems into human cells. The molecules landed on two neighboring targets within a single gene and snipped the DNA at both sites, cutting out the short stretch in between. The cell's own repair enzymes then grabbed the two sliced ends and stitched them back together. The procedure, in other words, surgically removed a piece of DNA, leaving no scar behind. And when the cell divided, its descendants inherited that deletion.

Before long, scientists were starting to use CRISPR to replace stretches of genes with new sequences. Along with the Cas9 enzymes and RNA guides, the researchers would deliver small pieces of DNA to cells. After the enzymes cut out a section of DNA, the cells would patch the new pieces into the gap.

CRISPR was a drastic improvement on both X-ray mutagenesis and restriction enzymes. CRISPR did not introduce random mutations like mutagenesis. Nor was it limited to inserting an existing gene from one species into another. Since researchers were now able to synthesize short pieces of DNA from scratch, CRISPR could potentially let them make any sort of change they wanted to any species' own genes.

In the 1970s,
Rudolf Jaenisch, a biologist at MIT, had used restriction enzymes to engineer mice for the first time. With the advent of CRISPR, he wondered if he could create new lines of mice with that tool as well. Collaborating with Feng Zhang, he and his graduate students and postdoctoral
researchers played around with CRISPR until they found a chemical recipe they could use to slip the molecules into a fertilized mouse egg. They were able to alter as many as five different genes at once by delivering five different RNA guides. Jaenisch and his colleagues then implanted these altered eggs in female mice, where they developed into healthy pups. Eighty percent of the time, Jaenisch's team successfully engineered precisely the changes they desired.

A new generation of graduate students silently thanked Jaenisch every day for making their lives easier. Many PhD projects had to start with the creation of a mouse model to study a gene or a disease. It typically took eighteen months to create a line of mice, and often it took more than one try to get the mouse right. Now, with CRISPR, Jaenisch needed only five months to get the job done.

I was working as a reporter during those frenzied years, and I did my best to keep up with CRISPR's advances. But very soon
the parade of CRISPR animals became a stampede. Scientists were altering the DNA of zebrafish and butterflies, of beagles and pigs. By 2014, it dawned on me that I was witnessing the beginning of something enormous. Biologists began speaking about their life before and after CRISPR. But I didn't truly appreciate what CRISPR meant to scientists until I returned to Cold Spring Harbor one early spring day to spend an afternoon in a giant greenhouse with cathedral-like ceilings made of glass.

A plant scientist named Zachary Lippman led me down narrow aisles past rows of pots, each with a plant climbing a tall stake. Although he was still young, Lippman had a pair of gray patches in the dark beard framing his chin. I wondered if the six children he and his wife were raising might have something to do with them. “They say I do genetics at the lab and then do genetics at home,” Lippman said.

Lippman has a long history of showing off his plants. Growing up in Connecticut, he worked on a farm where he learned how to grow giant pumpkins. At the peak of the growing season, they put on ten or fifteen
pounds a day. “
To me the interest was how the hell does this thing get so big, and how can I get it bigger?” Lippman said.

Heading to Cornell for college, Lippman majored in plant breeding and genetics. There he discovered that scientists had long been asking his boyhood question—not just about pumpkins but about other fruits and vegetables. One of the key changes to crops during the Agricultural Revolution was making them bigger—to turn the stubby fruits of teosinte into long ears of corn, to swell the thin pale roots of carrots into stout orange tubers.

Using traditional methods for studying genes, the scientists had found some of the mutations that made these changes possible. They had done a lot of this work on tomatoes, Lippman discovered, because their biology lends them well to genetic experiments. Lippman followed in their scientific footsteps by studying tomatoes, too.

“Look at these tiny little berries,” Lippman said to me. He had stopped at a tomato plant towering over us. Grabbing a stem, he cradled its fruit. “These plants here are the closest that we know to the first domesticated forms of tomato,” he said.

The domestication of tomatoes by the indigenous farmers of Peru turned blueberry-sized fruits into the larger kind we find in supermarkets and at farm stands. Lippman's research has helped reveal how those earlier breeders made tomatoes big. It turns out that they had to change the shape of tomato flowers.

When a bud on a tomato plant begins developing into a flower, it first divides up into wedges, called locules. From those locules will develop the petals of the tomato flower. And at the center of the flower, those same locules will give rise to the sections of a tomato. One gene controls how many locules form on a tomato plant. Mutate the gene, and the plant makes more locules. And more locules develop into a bigger tomato.

This locule-controlling gene was not the only one to mutate during the domestication of tomatoes. Lippman's research has also revealed that the crops adapted to the length of day as the crops were moved to different parts of the world.

Lippman and his colleagues found that wild tomatoes, which grow at the
equator in South America, are adapted to getting twelve hours of sunlight every day through the year. When they brought wild tomato plants from the Galápagos Islands north to Cold Spring Harbor, they discovered that the plants fared poorly, thanks to the long New York summer days. The plants responded to the extra sunlight with flower-suppressing proteins, delaying the growth of their fruits until the end of the season. But domesticated tomatoes that grow in Europe and North America have acquired mutations that caused the plants to make fewer anti-flowering proteins in the summer.

In 2013, Lippman learned that scientists had figured out how to use CRISPR to edit genes in a plant for the first time. He got hold of the molecules and tested them out on tomatoes to see how well they worked. No genetic tool he had used before came close. “It was black and white,” Lippman said. “We just sat down and had brainstorming sessions, just saying, ‘What can we do?'”

One of the first items on their list was to get tomatoes to stop making flower-suppressing proteins altogether in response to long summer days. They used CRISPR to cut out the genetic switch for this activity in domesticated tomatoes. When
Lippman and his colleagues planted the seeds of these altered plants, they grew their flowers—and their tomatoes—two weeks ahead of schedule. They might thrive in places with much shorter summers. “Now you can start to think about growing some of your best tomato varieties in even more northern latitudes, like in Canada,” Lippman said.

Lippman had, in effect, created a new crop variety in one step. He did not need the eye of Luther Burbank, scanning thousands of plants for a single promising mutant each year. Nor did he need to transfer a gene from some other species to create a genetically modified crop. He directly altered the tomato's own genes, using the knowledge he had gained about how tomatoes work.

This success made Lippman's brainstorming more ambitious. He wanted to turn a wild plant into a domesticated crop. And for his new experiments, he chose ground-cherries.

I couldn't really appreciate what he was doing, Lippman assured me, unless I ate some ground-cherries first. He brought me a plastic box filled with
golden fruits the size and shape of marbles. When I bit into a ground-cherry, I tasted a rich flavor that hovered somewhere between pineapple and orange. The fruits were so delicious and so distinctive that I wondered why I hadn't had one before. The reason, Lippman explained to me, is that they're wild.

Ground-cherries (known scientifically as
Physalis
) live across much of North and South America. They grow into bushes and develop their fruit inside a lantern-shaped husk. Native Americans gathered ground-cherries to make sauces, and European settlers followed suit. Some collected the seeds and planted them in their gardens. Today you can buy a packet of ground-cherry seeds, and sometimes you can find the fruits for sale at a farmers' market or a gourmet store. But because they're wild, ground-cherries remain an oddity rather than a crop. The fruits ripen one by one through a long season, and gardeners have to wait for them to drop to the ground before collecting them—hence their name.

Lippman has long had a scientific curiosity about ground-cherries, because they belong to the same family as tomatoes. Their close evolutionary relationship means that they have a lot of biology in common. Both ground-cherries and tomatoes form their flowers and fruits from locules, for example, and they use related versions of the same genes to build them. It's intriguing to Lippman that tomatoes were domesticated but their cousins, the ground-cherries, never were.

One possibility for this difference may be that ground-cherry DNA doesn't lend itself to easy domestication. Tomatoes, like humans, have two copies of each chromosome. But ground-cherries have four. To breed ground-cherries for some particular trait, farmers need to find plants that inherited the same mutation on all four copies of one of their genes. It occurred to Lippman that he could use CRISPR to edit mutations directly into ground-cherries instead.

Lippman scooted down an aisle, the leaves brushing against his shoulders. He found a ground-cherry bush that he had edited with CRISPR. It had flowered a few days before, and by now the petals had fallen off. The sepals—the leaflike petals that surround the flower—had expanded to form the papery lanterns inside which the fruit would now develop.

On an ordinary ground-cherry plant, these lanterns would have five sepals. Lippman peeled off the sepals on his edited plant, counting them as he went: “One, two, three, four, five, six, seven.”

Once he had pulled away all the sepals, Lippman revealed a tiny young ground-cherry fruit inside. It had seven locules now instead of the normal five.

“We could never do this with traditional breeding,” Lippman said. “And we got this”—he snapped his fingers—“in one generation. All four copies of the gene mutated.”

Lippman was soon going to test other edits. He would edit genes that controlled when the fruits fell from the bushes, so that farmers wouldn't have to rummage on the ground for them. He would make a change to get the plants to ripen their fruits in batches rather than a few at a time. He would adjust the plants' response to sunlight so they would start producing fruit early in the growing season. They would grow to a fixed height so that farmers could use machines to gather them.

Lippman planned on starting by editing plants for one trait at a time. If he succeeded, he would then create RNA guides that could alter all the traits at one shot, in one plant. When that ground-cherry plant reproduced, its offspring could inherit all the genetic machinery required to be a domesticated plant instead of a wild one.

“I know this sounds a little ridiculous,” Lippman confessed, “but I think this will be the next berry crop.” Having listened to his plan, I didn't think it was ridiculous at all. I thought Lippman was being too modest. He was trying to replay the Agricultural Revolution on fast-forward. Instead of a thousand years, he might only need a single growing season.

To CRISPR, ground-cherries and humans proved pretty much the same: Their DNA was equally easy to cut.

Scientists quickly began using CRISPR to edit the genes of human cells, to answer questions about ourselves that once seemed unanswerable. We each carry about twenty thousand protein-coding genes, and thousands
more genes that encode important RNA molecules. But how many of those do we really need? When mutations shut down some genes, it leads to lethal hereditary diseases. Yet many of us walk about in good health despite inheriting some broken genes. Scientists have long wondered just how many genes in the human genome are absolutely essential to our survival. But they knew it was impossible to actually compile that catalog.

CRISPR made it possible. In 2015, three separate teams of scientists used CRISPR to shut down all the protein-coding genes in human cells, one at a time, to see if the cells could survive without them. They ended up with lists that were pretty much identical.
About two thousand genes, only about 10 percent of all the protein-coding genes in the human genome, proved to be essential. The experiments showed that many genes were expendable because they had backup. If they failed, other genes could take over their jobs.

Other scientists began experimenting with CRISPR on human cells with a different goal: to invent new forms of medicine. In December 2013, a team of Dutch researchers demonstrated how CRISPR medicine might work. They took samples of
cells from people with cystic fibrosis and raised colonies of them in dishes. The cells all shared the same defective mutation in a gene called CFTR. The scientists fashioned CRISPR molecules that could chop out the mutation, and then they stitched a working version of that DNA in its place.

It soon became clear that CRISPR's power was not limited to altering somatic cells. It could change the DNA in germ-line cells as well. In December 2013, a group of scientists at the Shanghai Institutes for Biological Sciences in China reported the results of an experiment on
mice that suffered from hereditary cataracts. The scientists injected CRISPR molecules into mouse zygotes and they repaired the mutant gene. The altered mice grew up to be fertile adults, and their descendants gazed through clear eyes.