Medical & Biotech Matters: Every Cell in Your Body Has the Same DNA. Except It Doesn’t.

Medical & Biotech Matter
Every Cell in Your Body Has the Same DNA. Except It Doesn’t.

James Priest couldn’t make sense of it. He was examining the DNA of a desperately ill baby, searching for a genetic mutation that threatened to stop her heart. But the results looked as if they had come from two different infants.

“I was just flabbergasted,” said Dr. Priest, a pediatric cardiologist at Stanford University.

The baby, it turned out, carried a mixture of genetically distinct cells, a condition known as mosaicism. Some of her cells carried the deadly mutation, but others did not. They could have belonged to a healthy child.

We’re accustomed to thinking of our cells sharing an identical set of genes, faithfully copied ever since we were mere fertilized eggs. When we talk about our genome — all the DNA in our cells — we speak in the singular.

But over the course of decades, it has become clear that the genome doesn’t just vary from person to person. It also varies from cell to cell. The condition is not uncommon: We are all mosaics.

For some people, that can mean developing a serious disorder like a heart condition. But mosaicism also means that even healthy people are more different from one another than scientists had imagined.

Magical Mystery Tour

In medieval Europe, travelers making their way through forests sometimes encountered a terrifying tree.

A growth sprouting from the trunk looked as if it belonged to a different plant altogether. It formed a dense bundle of twigs, the sort that people might fashion into a broom.

Germans call it Hexenbesen: witches’ broom. As legend had it, witches used magic spells to conjure the brooms to fly across the night sky. The witches used some as nests, too, leaving them for hobgoblins to sleep in.

In the 19th century, plant breeders found that if they cut witches’ broom from one tree and grafted it to another, the broom would grow and produce seeds. Those seeds would sprout into witches’ broom as well.

Today you can see examples of witches’ broom on ordinary suburban lawns. Dwarf Alberta spruce is a landscaping favorite, growing up to ten feet high. It comes from northern Canada, where botanists in 1903 discovered the first known dwarf clinging to a white spruce — a species that can grow ten stories tall.

Pink grapefruits arose in much the same way. A Florida farmer noticed an odd branch on a Walters grapefruit tree. These normally bear white fruit, but this branch was weighed down with grapefruits that had pink flesh. Those seeds have produced pink grapefruit trees ever since.

Charles Darwin was fascinated by such oddities. He marveled at reports of “bud sports,” strange, atypical blooms on flowering plants. Darwin thought they held clues to the mysteries of heredity.

The cells of plants and animals, he reasoned, must contain “particles” that determined their color, shape and other traits. When they divided, the new cells must inherit those particles.

Something must scramble that heritable material when bud sports arose, Darwin declared, like “the spark which ignites a mass of combustible matter.”

Only in the 20th century did it become clear that this combustible matter was DNA. After one cell mutates, scientists found, all its descendants inherit that mutation.

Witches’ broom and bud sports eventually came to be known as mosaics, after the artworks made up of tiny tiles. Nature creates its mosaics from cells instead of tiles, in a rainbow of different genetic profiles.

Before DNA sequencing was commonplace, scientists struggled to tell the genetic differences between human cells. Cancer offered the first clear evidence that humans, like plants, could become mosaics.

In the late 1800s, biologists studying cancer cells noticed that many of them had oddly shaped chromosomes. A German researcher, Theodor Boveri, speculated at the turn of the century that gaining abnormal chromosomes could actually make a cell cancerous.

As soon as Boveri floated his theory, he faced intense opposition. “The skepticism with which my ideas were met when I discussed them with investigators who act as judges in this area induced me to abandon the project,” he later said.

Boveri died in 1915, and it took nearly five decades for scientists to discover he was right.

David A. Hungerford and Peter Nowell found that people with a form of cancer called chronic myelogenous leukemia were missing a substantial chunk of chromosome 22. It turned out a mutation had moved that chunk over to chromosome 9. The cells that inherited that mutation became cancerous.

It’s hard to think that a tumor might have anything in common with a pink grapefruit. Yet they are both products of the same process: lineages of cells that gain new mutations not found in the rest of the body.

Some skin diseases proved to be caused by mosaicism, too. Certain genetic mutations cause one side of the body to become entirely dark. Other mutations draw streaks across the skin.

The difference is in the timing. If a cell gains a mutation very early in development, it will produce many daughter cells that will end up spreading across much of the body. Late-arising mutations will have a more limited legacy.

A Brain Biography

Dr. Walsh and his colleagues have found evidence of mosaicism in some very unexpected places.

They investigated a mysterious disorder called hemimegalencephaly, which causes one side of the brain to become overgrown. The researchers examined tissue from patients who had brain surgery to treat the seizures triggered by hemimegalencephaly.

Some of the brain cells in the patients — but not all of the cells — shared the same mutant genes. It’s possible that these mutant neurons multiplied faster than others in the brain, triggering one side to become enlarged.

Preliminary studies suggest that mosaicism underlies many other diseases. Last year, Christopher Walsh, a geneticist at Harvard University, and his colleagues published evidence that mosaic mutations may raise the risk of autism.

But scientists are also finding that mosaicism does not automatically equal disease. In fact, it’s the norm.

When a fertilized egg — known as a zygote — starts dividing in the womb, many of its early descendant cells end up with the wrong number of chromosomes. Some are accidentally duplicated, and others lost.

Most of these unbalanced cells divide only slowly or die off altogether, while the normal cells multiply far faster. But a surprising number of embryos survive with some variety in their chromosomes.

Markus Grompe, a biologist at Oregon Health & Science University, and his colleagues looked at liver cells from children and adults without liver disease. Between a quarter and a half of the cells were aneuploids, typically missing one copy of one chromosome.

Along with altered chromosomes, human embryos also gain smaller mutations in the genome. Stretches of DNA may be copied or deleted. Single genetic letters may get incorrectly reproduced.

It wasn’t possible to study such molecular changes accurately until DNA-sequencing technology became sophisticated enough.

In 2017, researchers at the Wellcome Trust Sanger Institute in England examined 241 women, sequencing batches of white blood cells from each. Every woman had acquired about 160 new mutations, each present in a sizable fraction of her cells.

The women gained these mutations as embryos, the scientists theorized, with two or three new mutations arising each time a cell divided. As those new mutations occurred, the embryonic cells passed them all down to their descendants, a mosaic legacy.

Dr. Walsh and his colleagues have discovered intricate mosaics in the brains of healthy people. In one study, they plucked neurons from the brain of a 17-year-old boy who had died in a car accident. They sequenced the DNA in each neuron and compared it to the DNA in cells from the boy’s liver, heart and lungs.

Every neuron, the researchers found, had hundreds of mutations not found in the other organs. But many of the mutations were shared only by some of the other neurons.

It occurred to Dr. Walsh that he could use the mutations to reconstruct the cell lineages — to learn how they had originated. The researchers used the patterns to draw a sort of genealogy, linking each neuron first to its close cousins and then its more distant relatives.

When they had finished, the scientists found that the cells belonged to five main lineages. The cells in each lineage all inherited the same distinctive mosaic signature.

Even stranger, the scientists found cells in the boy’s heart with the same signature of mutations found in some brain neurons. Other lineages included cells from other organs.

Based on these results, the researchers pieced together a biography of the boy’s brain.

When he was just an embryonic ball in the womb, five lineages of cells had emerged, each with a distinct set of mutations. Cells from those lineages migrated in different directions, eventually helping to produce different organs — including the brain.

The cells that became the brain turned into neurons, but they did not all belong to the same family. Different lineages merged together. In essence, the boy’s brain was made of millions of mosaic clusters, each composed of tiny cellular cousins.

It’s hard to say what these mosaic neurons mean to our lives — what it means for each of us to have witches’ broom growing in our skulls. “We don’t know yet whether they have any effect on shaping our abilities or challenges,” said Dr. Walsh.

What we do know is that mosaicism introduces randomness into the development of our brains. Mutations, which arise at random, will form different patterns in different people. “The same zygote would never develop exactly the same way twice,” said Dr. Walsh.

A Heart in Pieces

As ubiquitous as mosaicism may be, it’s still easy to overlook — and surprisingly hard to document.

Astrea Li, the infant examined by Dr. Priest at Stanford, had gone into cardiac arrest the day she was born. Her doctors put a defibrillator in her heart to shock it back into the proper rhythm.

Dr. Priest sequenced Astrea’s genome to search for the cause of her disorder. He concluded that she had a mutation in one copy of a gene called SCN5A. That mutation could have caused her trouble, because it encodes a protein that helps trigger heartbeats.

But when Dr. Priest ran a different test, he couldn’t find the mutation.

To get to the bottom of this mystery, he teamed up with Steven Quake, a Stanford biologist who had pioneered methods for sequencing the genomes of individual cells. Dr. Priest plucked 36 white blood cells from the child’s blood, and the scientists sequenced the entire genome of each cell.

In 33 of the cells, both copies of a gene called SCN5A were normal. But in the other three cells, the researchers found a mutation on one copy of the gene. Astrea had mosaic blood.

Her saliva and urine also turned out to contain mosaic cells, some of which carried the mutation. These findings demonstrated that Astrea had become a mosaic very early in her development.

The skin cells in her saliva, the bladder cells in her urine and her blood cells each originated from a different layer of cells in two-week-old embryos.

Astrea’s SCN5A mutation must have originated in a cell that existed before that stage. Its daughter cells later ended up in those three layers, and ultimately in tissues scattered throughout her body.

They might very well have ended up in her heart, too. And there the mutation could have theoretically caused Astrea’s problems.

While Dr. Priest was reconstructing Astrea’s mosaic origins, she was recovering from the surgery to implant her defibrillator. Her parents, Edison Li and Sici Tsoi, brought her home. And for a few months, it seemed she was out of the woods.

One day, however, her defibrillator sensed an irregular heartbeat and released a shock — along with a wireless message to Astrea’s doctors.

Back at the hospital, doctors discovered a new problem: her heart had become dangerously enlarged. Researchers have linked mutations in the SCN5A gene to the condition.

Her heart soon stopped. Her doctors attached a mechanical pump, and soon a donated heart became available.

Astrea underwent transplantation surgery and recovered well enough to go home. She went on to enjoy a normal childhood, performing cartwheels with her sister and listening obsessively to the soundtrack of “Frozen.”

The transplant did not just give Astrea a new lease on life. It also gave Dr. Priest a very rare chance to look at a mosaic heart up close.

The transplant surgeons had clipped some pieces of Astrea’s cardiac muscle. Dr. Priest and his colleagues extracted the SCN5A gene from the cells taken from different parts of her heart.

On the right side of the heart, he and his colleagues found that more than 5 percent of the cells had mutant genes. On the left, nearly 12 percent did.

To study the effect of this mosaicism, Dr. Priest and his colleagues built a computer simulation of Astrea’s heart. They programmed it with grains of mutant cells and let it beat.

The simulated heart thumped irregularly, in much the same way Astrea’s had.

The experience left Dr. Priest wondering how many more people might be at risk from a hidden mix of mutations.

Unless he winds up with another patient like Astrea, we may never find out.

Hacking DNA: The Story of CRISPR, Ken Thompson, and the Gene Drive

The very nature of the human race is about to change. This change will be radical and rapid beyond anything in our species’ history. A chapter of our story just ended and the next chapter has begun.

This revolution in what it means to be human will be enabled by a new genetic technology that goes by the innocuous sounding name CRISPR, pronounced “crisper”. Many readers will already have seen this term in the news, and can expect much more of it in the mainstream media soon. CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats and is to genomics what vi (Unix’s visual text editor) is to software. It is an editing technology which gives unprecedented power to genetic engineers: it turns them into genetic hackers. Before CRISPR, genetic engineering was slow, expensive, and inaccurate. With CRISPR, genome editing is cheap, accurate, and repeatable.

This essay is a very non-technical version of the CRISPR story concluding with a discussion of Gene Drive1, a biological technique which, when used with CRISPR, gives even greater power to genetic engineers. The technical details go very deep and for those who are interested in diving in, I’ve included a number of useful pointers. At the end, I will very briefly discuss the implications of these two new technologies.

First, a bit of background. The genetic code described in DNA can be thought of as the software that builds every life form on our planet. Geneticists are learning to decode genomes, but have been hobbled by the fact that they have very limited ability to modify that code. It is as if software engineers have access to vast numbers of supremely powerful programs which they barely understand and cannot edit at all. But given the ability to edit and change the genetic software, learning can begin in earnest. Not only is it far easier to discover how the code works, but bugs can be fixed and improvements made; hackers will now emerge.

It is easiest to understand this amazing technology by learning where it came from. In the 1980s Japanese researchers at Osaka University were sequencing the DNA of the bacteria E. Coli when they noticed something weird. DNA is built of long strands of nucleotides and within the bacterial DNA were repeated clusters of strange, out-of-place nucleotide sequences that were themselves interrupted by seemingly random strands of DNA. Over time, similar clusters of repeated nucleotide sequences were found in several other bacteria and were christened CRISPR because, well, the term pretty much described the  sequences the researchers found. But no one knew at the time why they were there, nor their function.

There were several discoveries leading to the answer to these questions but two were critical. First, three different teams used newly available databases of genetic material to point out that those weird CRISPR sequences in bacteria looked a whole lot like vastly-out-of-place viral DNA. Second, a brilliant evolutionary biologist at the National Center for Biotechnology Information, Eugene Koonin, had the critical insight that this was a bacteriological defense mechanism against viruses. Eventually, this idea led to the discovery of CRISPR’s purpose.

We humans fight our own, all-too-frequent battles with both bacteria and viruses, and thus it may come as a bit of a surprise to learn that bacteria and viruses have been locked in their own epic battle for survival for billions of years. Every day trillions upon trillions of bacteria are killed by viruses (a virus which attacks bacteria is called a “phage”). Many more manage to fend off their phage foes, largely thanks to various armaments the bacteria have evolved over the eons – a rudimentary form of immune system. CRISPR, it turns out, is a highly effective weapon in this bacterial immune system.

First, in order to avoid confusion, let me just clarify one thing. As we saw, “CRISPR” was originally a description of certain weird DNA sequences. To use those sequences as a defense mechanism, or, as we shall see, to create an edit, a tool called Cas, for CRISPR-associated system, is needed. Sometimes, when referring to a specific type of Cas protein you will also see Cas9 (or Cas3, etc.). The complete toolset is therefore sometimes called CRISPR/Cas together, but is usually just abbreviated to CRISPR, which is the convention I am following.

Here’s how it works. When certain bacteria with CRISPR in their arsenal manage to fight off a viral attack, they then use the Cas enzymes to grab fragments of the viral DNA and insert them into their own DNA. If the same virus makes the mistake of attacking such a well-prepared bacteria, the bacteria would use the CRISPR sequence as a template to recognize the virus, then employ the Cas enzyme to chop it up into inert, non-threatening pieces.

It’s a very cool mechanism. Cas enzymes copy the CRISPR viral genes from their own DNA into an RNA molecule (like DNA, RNA is fundamental to gene expression) creating a Cas plus RNA package. It then take a random trip around the bacterial cell. When the Cas package bumps into another molecule it checks for DNA and if found, reads the nucleotide sequence. If it matches the copied CRISPR, we have a matching virus. The RNA then latches on and the Cas enzymes act like a guillotine, chopping up the viral DNA and rendering it inactive (or “dead” if one wants to believe a virus was ever alive). If this sounds to you sort of like an immunology algorithm realized in biology, you would be right:

    CRISPR.wander() – until encounter another molecule
        If (new molecule contains DNA and DNA matches
            CRISPR.dna) CRISPR.cas9.grab() // nab the viral dna
            CRISPR.cas9.chop() // kill the virus!
    continue

Cool, right? Decoding this mystery might have remained worthy of a path to a doctorate or from associate to full professor, instead of the Nobel-worthy, humanity-altering discovery it has become, if it hadn’t been for the insights of a several different (and now competing2) teams, each of whom had the same brilliant insight. They understood that this eons-old defense system had the potential to revolutionize genetic engineering. If the CRISPR mechanism could be harnessed, they would have a tool that could precisely chop out a piece of DNA. What’s more, with CRISPR and two different Cas9 enzymes, they could precisely chop out an entire segment of arbitrary composition. This is only half of an edit, of course. Once you remove the unwanted sequence you still need to replace it with the gene you wish in its place. As it turns out, this is a piece of cake. It is sufficient to simply inject the gene(s) you want to replace the snipped segments, and already-existing DNA repair enzymes will put everything back together.

One of the coolest features of CRISPR is that unlike most of the existing gene editing techniques, which only work on a limited number of organisms, CRISPR is generic. It will, at least in theory, work in any type of cell in any (earthly) life form.

Let’s just stop for a minute and contemplate how amazing CRISPR may just be for the future of humanity. We will be able to neutralize enemies in the animal kingdom. We will be able to cure genetic diseases. We may be able to develop therapies against any viral or bacteriological foe that will be custom and perfectly effective. There may even be a cure for cancer. And (or perhaps I should say “But”) we will also have the capacity to build custom designed human beings or, for that matter, any other life form.

So that is CRISPR: the most powerful genetic engineering tool ever created. But CRISPR only allows us to modify one gene at a time, one organism at a time. To make species-level changes, CRISPR must be amplified by another powerful phenomenon: gene drive. First, a definition and then a brief but relevant diversion to one of the most famous programming hacks ever.

The concept of gene drive has been around for almost 15 years. Particular versions of genes (aka alleles) promulgate themselves through generations in sexually reproducing species, with 50% odds of that allele being inherited by each offspring (assuming only one parent has that version), since ½ of the DNA comes from each parent. So, if you have one allele for blue eyes and one allele for brown eyes, your child has a 50% chance of getting either. Obviously, outside of any external force this means in the next generation the allele will have a 25% chance and so on. What might that external force be? The most obvious is natural selection. If a particular allele confers an advantage to its owner, then that allele is more likely to promulgate through a population. Gene drive is any mechanism that makes a gene particularly “selfish” in that it increases the probability that that particular gene will be inherited above 50%, regardless of any selection pressure.

This is important because if you want to use CRISPR to have a big impact on populations, say to change the Anopheles mosquito to no longer carry malaria, instead of having to CRISPRize millions of mosquitos and still see an attenuation in future generations (assuming the change does not confer an advantage to the mosquito) then a gene drive which placed the genes in the population more rapidly would have a huge advantage. Here is where a new character in the story pops up, Kevin Esvelt at MIT. Esvelt’s idea was to create a gene drive using the unparalleled editing accuracy of CRISPR. In doing so, he created the most powerful gene drive ever.

To understand what he did, let’s take a brief tangent into the world of software. In 1984, Ken Thompson, the inventor of the Unix operating system, and one of the greatest programmers of all time, wrote about his favorite hack3. It was a brilliant, recursive idea in which he created a virus capable of infecting the core infrastructure of every Unix operating system. It worked like this.

• First Thompson modified the source code (written in the C language) for the Unix “login” program to give himself a secret backdoor to become any user on the system.

if (password == “user’s password” or password == “ken’s special password”)
Log user in.

At this point his relatively modest hack would have given Thompson complete access to any Unix system which included his compiled version of the login code. But it would have also been completely obvious to anyone who looked at the login program.

• Next (and this is the truly insidious aspect of the hack), Thompson modified the source code of the C language compiler to recognize when it was compiling the login program code and to inject the viral code into its compiled output, regardless of the original source.4

Thus, the core code that rebuilds Unix was, itself, changed to sneakily create a hacked version of Unix. This is actually a pretty good analogue to Kevin Esvelt’s DNA hack.

Esvelt realized that DNA is the compiler of life itself. Everything that life did, including CRISPR, was created by that compiler. So, like Thompson, Esvelt realized that one could modify the compiler itself to change what it was compiling, regardless of the instructions within the program. So, here is a simplified explanation of Esvelt’s hack:

• Create a CRISPR edit, which will CRISPRize embryonic DNA with a desired change. For example, replace a brown eye color gene with a blue eye color gene.
• Create a separate CRISPR edit, which will embed the instructions required to create the first CRISPR edit within that same DNA. In other words, the compiler of life has now been modified to recognize and modify other DNA.
• During fertilization, when the modified DNA meets its new pair, those instructions will cause the creation of a CRISPR tool which will then CRISPRize that new DNA ensuring both strands of the pair have the desired change. Thus, even if the DNA from the non-CRISPRized parent had a brown eye color gene, that gene will now be changed to blue.
• The resulting organism, and all of its offspring, will then also contain the change. So long brown eyes!

This is the ultimate gene drive. Whatever modification you create can then be replicated in every descendent with nearly 100% reliability5. This will cause the new trait to flow through a species’ genome with unprecedented speed.

There will be many applications of gene drive as we attempt to control or manage various organisms, from mosquitos to bacteria. However, let’s take a moment to imagine the applications in our own species. One might argue that it is immoral to modify human embryos in this fashion and that politicians, religious leaders, and ethicists will outlaw using CRISPR and, especially, gene drive to change humanity. But on the other hand, think of the benefits. Gene-related disease can be eliminated entirely, before a child is even born. What’s more, as our understanding of the genome improves, think of the advantages we might confer on our children, and with gene drive, our children’s children. And think about the advantages to a society that can, for example, raise all children’s intelligence by 10 (or 20 or 30) IQ points.

What will stop people from attempting to drive desirable characteristics into a population? Continuing the example above, what happens if and when scientists develop a solid understanding of the genetic underpinnings of advanced intelligence? What will stop a government from mandating those changes in their population? And what will competing governments then choose to do?

We have already begun to fund CRISPR based companies at Y Combinator, and I expect we will fund many more. It is important to understand that once genetic programmers have access to life’s code, the sky’s the limit. Already the variety of applications, real and proposed, is stunning. Scientists have used CRISPR to modify goats to produce spider silk, an extraordinary material but very difficult to make in quantity, in their milk! It has been proposed that CRISPR can cure hemophilia and replace antibiotics. And Chinese scientists have controversially used CRISPR to modify (non-viable) human embryos. CRISPR-enhanced humans are not as distant as you might think.

CRISPR techniques are getting better and better. More accurate. More predictable. Cheaper. And we are learning more and more about the genetic code (partially thanks to our ability, now using CRISPR, to see what happens when we poke out one gene and replace it with another). The trends are unstoppable and the conclusion unavoidable: in the not very distant future we will be able to program most any animal in most any way we wish, including human beings. Whether we will resist the urge to tinker with what it means to be human is an open question, but I predict it’s only a matter of time until someone or some society will take that plunge.

Mail-Order CRISPR Kits Allow Absolutely Anyone to Hack DNA

Experts debate what amateur scientists could accomplish with the powerful DNA editing tool—and whether its ready availability is cause for concern

Mail-Order CRISPR Kits Allow Absolutely Anyone to Hack DNA
abd-A mutant hatchling created by CRISPR-Cas9. Credit: Erin Jarvis and Nipam Patel, UC Berkeley

“We aren’t going to get sick, are we?” my roommate Brett asked me. He cringed as I knelt down and stuffed a plate of E. coli bacteria—which came as part of the DIY CRISPR–Cas9 kit I bought online—into our fridge next to cartons of eggs, strawberry jam, bottles of beer and a block of cheese.

“No, we won’t. The label says ‘non-pathogenic,’” I replied, trying to sound assuring. But honestly, I had no clue what I was doing. I nudged all the food up against the fridge wall, and left a two-inch border around the plate of living cells—a no man’s land between the microbes and our dinner. A couple inches probably would not stop the bugs, but I figured it couldn’t hurt.

CRISPR–Cas9 (or CRISPR, for short) has given scientists a powerful way to make precise changes to DNA—in microbes, plants, mice, dogs and even in human cells. The technique may help researchers engineer drought-resistance crops, develop better drugs, cure genetic disorders, eradicate infectious diseases and much more. Ask any biologist, and they’ll likely tell you that CRISPR is revolutionary. It’s cheap and effective, and in many cases, it works much better than older methods for making genetic modifications. Biologists will also tell you that CRISPR is very easy to use. But what does “easy to use” mean?

I am not a DIY scientist, much less a professional scientist. You won’t find me swabbing my cheek cells for DNA or tinkering with yeast in a lab on the weekend. But I wondered: Is CRISPR so easy that even amateurs like me can make meaningful contributions to science? And also, does this new technique make gene editing so accessible that we need to worry about DIY scientists cooking up pandemic viruses in their basements? If you Google ‘DIY CRISPR,’ stories such as “What Happens If Someone Uses this DIY Gene Hacking Kit to Make Mutant Bacteria?” pop up.

I attempted to find answers to all these questions myself, starting with the plate of bacteria in the kitchen of my San Francisco apartment.

CUT AND SPLICE

CRISPR stands for “clustered regularly interspaced short palindromic repeats.” The CRISPR system is made up of two components: a protein called Cas9 and a guide RNA, a string of nucleic acid molecules with a certain genetic code. Put them together, and they create a tool you can use to tweak an organism’s genome. To do this, CRISPR searches the organism’s DNA for a certain sequence—specifically, the one encoded by the guide RNA, which holds the inverse sequence of your target DNA. “Cas9 opens up the DNA, it separates the strands of the double helix in a very small area, and allows the guide RNA to pair with one of the strands,” explains Dana Carroll, a professor of biochemistry at the University of Utah. “If it is a good match, cutting occurs. If it is not a good match, Cas9 and [the] guide RNA fall off and try again somewhere else.” When it finds the right sequence, the Cas9 protein slices the DNA at that precise spot.

At this point, if you leave the cell alone, it will usually mend CRISPR’s cut—but it will occasionally also make a mistake in the repair process, breaking a gene or other parts of the genome. Since CRISPR repeatedly goes back and slices the DNA again after the cell mends it, the gene eventually breaks, or, in technical terms, gets knocked out. And, if you add new DNA, the cell may incorporate it while fixing the cut. This means you can insert DNA where you want to in the genome—you just need to know the organism’s genetic sequence of your desired target area.

Scientists originally discovered this sophisticated system in archaea and bacteria, which deploy CRISPR to chop up invading viruses. But a few years ago, researchers figured out how to repurpose CRISPR to run in pretty much any living thing they want. And now it’s made genetic engineering easier than ever before.

For my own experiment, everything I needed came in a small cardboard box—an assortment of bottles, tubes, plates, powders and liquids (plus E. coli). I ordered my kit for $130 from the crowd-funding site Indiegogo as part of a campaign created by Bay Area biohacker Josiah Zayner. Zayner has a PhD in molecular biophysics and spent two years as a research fellow at NASA. He ran the crowdfunding campaign out of his apartment, and by the end of it, he had raised over $70,000 and sold 250 DIY CRISPR kits—one of which now sat on my kitchen table. Zayner has now sold over a thousand kits, largely on his company’s website, The Odin.

The idea behind the kit experiment is quite simple. The goal: modify the E. coli so that it can grow on an antibiotic called streptomycin, which normally kills bacteria. With materials and instructions from the kit, I will introduce CRISPR into the bacteria cells, and use it to rewrite a tiny part of their DNA, creating genetically altered cells that happily thrive on streptomycin. In the end, CRISPR will track down and then change only a single base pair (which are the building blocks for DNA) out of the 4.6 million base pairs in the E. coli genome. It will swap out the chemical compound adenine for cytosine—or, in terms of the genetic alphabet, an “A” for a “C.” Because of that tiny code change, my bacteria cells will make the amino acid lysine instead of another one, threonine. If my gene editing succeeds, this will stop streptomycin from interfering with the E. coli.

On a Monday afternoon in May, I donned latex gloves and spread newspaper over the dinner table. I grabbed three small plastic tubes from the freezer, picked up a pipette—a hand-held instrument, used in labs to measure out liquids—from my DIY kit, and started adding the CRISPR ingredients to tubes of E. coli.

I put in the Cas9 protein and guide RNA, which came as liquids in small, plastic test tubes, to my bacteria cells. Then I dipped my pipette into a small tube of DNA and tried to suck up 10 microliters. Nothing came out. I squinted at the tiny drop of clear liquid, and realized it had frozen solid. I had no clue why. Uh oh, I hope that’s not a problem. Would it harm my experiment? I had absolutely no idea, so I just waited for the DNA solution to thaw and then squirted it into the bacteria tube. After several more steps, I spread my CRISPR’d bacteria onto three plastic plates and put them in my laundry room. The instructions said to wait 24–48 hours, then check for small white dots of bacteria. If I saw dots, CRISPR had done its job of splicing in the streptomycin-resistance gene. If not … well, failure is also part of the scientific process.

I had no problem conducting the experiment—CRISPR is easy, I concluded. I basically just measured, scraped and stirred a bunch of ingredients, occasionally cooling them or heating them up. But for all the godlike powers that I imagined CRISPR gave me, I actually had little say over what I did to my bacteria. Everything was predetermined, with instructions laid out for me like steps in a cookbook: “Add 100 microliters Transformation mix to a new centrifuge tube,” “Incubate this tube in the fridge for 30 minutes,” and so on. Ultimately, I had made zero decisions. Of course, I could have designed a custom-made CRISPR experiment—but it would have taken more time, more materials, more money, and a lot more knowledge than I currently had.

I inspected my bacteria 48 hours later. Crossing my fingers, I lifted the lid on the first plate. No white dots. Then the second plate: nothing. My stomach sank with disappointment. Then I gently raised the lid off the third plate, and saw … something. The plate had two faint milky white circles. Had CRISPR worked? Maybe. But then why did one plate have white spots, but not the other plates? I’d followed the same steps for each of them. Maybe my mind was playing tricks on me. Or perhaps I had contaminated the third plate. If only I could show my plates to someone who knew how to interpret them. Like a scientist. Unfortunately, there was no one to ask in my kitchen.

OPEN TO EVERYONE

A few weeks later, I drove 40 miles south of San Francisco to meet a DIY scientist named Johan Sosa, who knows way more about CRISPR than I do. We met at BioCurious, a community laboratory in Sunnyvale where he works most weekends and some evenings. Located in Santa Clara, BioCurious is a co-working space outfitted with science equipment and shared by “scientists, technologists, entrepreneurs, and amateurs who believe that innovations in biology should be accessible, affordable, and open to everyone,” according to its Web site. The lab is funded by donations and members—Sosa is one of its several dozen members.

At six foot, five inches, Sosa towers over most people. “I’m probably the tallest DIY biologist,” he jokes. He laughs easily, which offsets his height to lend him a gentle, laid-back manner. He is 40 years old, his dark hair flecked with silver. Originally from Sri Lanka, Sosa came to the U.S. for college at 15 years old to study computer science, and he’s worked for Bank of America and IBM as a computer security specialist and software engineer. He now has a day job in computer security—but he spends most of his free time at BioCurious. “You could say I have no life,” he chuckles, “This is my biggest hobby.” He has learned everything he knows about science (both theory and lab techniques) from others at BioCurious, by reading science papers, watching YouTube videos, attending lectures, and also through trial-and-error in his own research.

Sosa is one of a few DIY scientists at BioCurious using CRISPR. He first read about the technique in 2012, from one of biochemist Jennifer Doudna’s papers in the journal Science. Doudna, a professor at University of California, Berkeley, is one of the pioneers of CRISPR. “I didn’t think it was that big of a deal, because I knew there were already other ways of modifying DNA,” he recalls, “But I did think, ‘This is something I could do.’” He started experimenting with CRISPR in 2013.

On a muggy, overcast afternoon, I followed Sosa inside BioCurious. We strolled through a lobby and into a large windowless room. It had big cabinets crammed with bottles of liquid, racks of latex gloves, a giant bio-hood, microwave and fridge. Microscopes, scales, centrifuges and a bunch of other well-worn science equipment lay scattered across lab desks. A calm thrum of buzzing machinery filled the room, and test tubes shook quietly in an incubator nearby. Johan walked around the room, searching for a thermometer. “One thing about a DIY lab is that you leave something somewhere, and it always ends up somewhere else,” he told me.
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I had joined Sosa at BioCurious so I could learn more about what CRISPR means for DIY scientists, and also to do a (hopefully) more successful experiment, with his help. We decided on a very basic goal: we would use the powerful editing tool to cut DNA he had already extracted from yeast cells. This task is easier than the one I tried in my kitchen, because you don’t need to get CRISPR inside cells in order to slice the DNA. Professional scientists might use such a method as an intermediate step, such as when they need to cut and paste DNA together to make a gene as part of a bigger research project. “That’s a type of experiment everyone does every day,” explains Charles Gersbach, a professor of biomedical engineering at Duke University—though he notes that traditionally, researchers have used a type of protein called a restriction enzyme, not CRISPR, to do this.

Sosa and I pulled on latex gloves and carefully pipetted liquids into tubes to make our guide RNA from scratch—we first synthesized DNA strands with a specific sequence we wanted, used that as a template for the RNA, then destroyed the DNA and isolated the guide RNA from our test tube mixture. Later, we put the RNA in a new test tube, along with the other materials needed to make CRISPR work in this experiment: protein buffer, bovine serum albumin (a protein isolated from cows), water. Sosa sucked up the yeast DNA in his pipette. Without warning, the plastic needle-nose tip fell off into the tube of DNA. Someone, he told me, had donated the tips to their lab, but they weren’t the correct size. “I guess you got the full DIY experience,” he smiled, and pushed the plastic tip back onto the pipette. Then he picked up the Cas9 protein. “Here’s the world-famous Cas9,” he said, and handed it to me. I added it to our test tube.

Until CRISPR, DIY scientists didn’t have an easy, cheap or reliable way to precisely edit DNA. Many of them couldn’t afford the pricey and imperfect tools that professional scientists used for gene editing at the time. “Before CRISPR, there was TALENS [transcription activator-like effector nucleases] and zinc finger nucleases—older technologies that were not as precise or reliable,” explains Sosa. “They were out of the budget and the time constraints of DIY scientists.” Sosa says that if a DIY-er used those other technologies, it might cost him or her thousands of dollars to do a genetic engineering experiment. But with CRISPR, it’s vastly more affordable, especially if you want to attempt an experiment more than once. “With TALENS, you try it once and fail,” says Sosa. “With CRISPR, you can try it multiple times. That alone is a big deal.”

This means that CRISPR gives DIY-ers a whole new way to do science. So far, Sosa and his lab mates have tried out CRISPR in a number of ways: cutting yeast genomes, slicing DNA inside E. coli cells, and attempting to modify the CRISPR system by shrinking it or attaching other molecules to it. Sosa has goals for his CRISPR research. “I want to understand how a cell really functions, and what are all the little things that happen in it,” he explains. “And when something goes wrong [such as in diseases], how to fix it or make it do what I want.”

After several hours, Sosa and I checked to see if CRISPR had cut our yeast DNA. We dyed our DNA-CRISPR mixture blue and ran it through an electrically charged gel, which separates bigger DNA pieces from smaller ones. Tiny channels in the gel run from one charged end to the other, and the sliced DNA strands are pulled through them towards the positively charged side. If our experiment succeeded, we should see two blue bands for the short CRISPR-cut DNA strands in one spot, and one blue band for a longer, uncut piece of DNA (our control) in another location.

Sosa carried the gel into the bathroom, where we turned off the lights and looked at it under blue light. I held my breath while I inspected the gel for markings. One light-blue band gleamed in the dark—the control—and another single band lit up the spot where we should have seen our CRISPR’d DNA. “I don’t know what happened, but it doesn’t look right,” Sosa said, “I don’t think it worked.”

I left the lab feeling defeated, and headed back to San Francisco. Sosa texted me a few minutes later.

Hey, I figured out what happened. There was no DNA to start with, he wrote.

What happened? I texted back.

I think the DNA had either degraded or gotten too diluted, he wrote.

Even if we had got all the other parts (RNA, proteins, etc.) working, it didn’t matter. We hadn’t given CRISPR any DNA to cut. My second attempt at CRISPR had utterly failed.

BETTER, FASTER, CHEAPER

My own frustrating struggles with CRISPR aside, I wanted to see what professional biologists are doing with CRISPR, so I visited the lab of Nipam Patel at the University of California, Berkeley. After a quick tour of the lab, I sat down and stared into the microscope at a small, writhing marine creature: Parhyale hawaiensis, commonly called a beach hopper. At one centimeter long, Parhyale looks puny—you’d step on it at the beach without even noticing. But under the microscope, this female hopper resembled a giant translucent shrimp with many powerful, kicking legs. Parhyale is the star of this lab. “We’re looking at how you develop an individual body,” explains Erin Jarvis, a PhD student in Patel’s lab, “And also how you build a body form over evolutionary time.” And they’re using CRISPR to do it.

With CRISPR, these researchers knock out so-called Hox genes in Parhyale. Hox genes are found in all animals, including humans, and they control the development of their body plans. Among other things, they determine what appendages—such as swimming legs, claws and antennae—grown on which section of the body. Knock out a certain Hox gene with CRISPR and Parhyale will grow forward-walking legs where it should have jumping legs, for example.

Parhyale has nine Hox genes, and Patel’s team has knocked out seven of them. The researchers also have plans to add completely new genes to Parhyale using CRISPR—they’ve already done it once, by inserting a gene that codes for green fluorescent proteins, which allowed the researchers to visualize where a specific Hox gene is expressed in Parhyale. “From an evolutionary perspective, this [gives] us insight into how body plans evolve between species,” when comparing Parhyale to, for example, the well-studied fruit fly, Drosophila, explains Patel. “We believe that such evolutionary patterns help us understand the general mechanisms by which evolution creates animal diversity.… What we learn improves our knowledge about the function of these genes in other animals, including humans.”

CRISPR has transformed how Patel and his colleagues do their research. His lab has looked at Parhyale for about 20 years now. Before CRISPR they used another technique to knock out genes that required a lot more money, and even then, it wasn’t very efficient. It cost them about $900 to knock out a single gene in a group of Parhyale embryos with their other method. The technique, called “RNA interference,” silences expression of a gene—it doesn’t genetically knock it out as CRISPR does. The problem was, sometimes the method didn’t work at all.

Now it costs them less than $100 to knock out a gene. “Suddenly, with CRISPR, you don’t have to decide, ‘Which one gene do I want to put all my resources into?’” Jarvis says, “You can try a lot of different genes.” And with CRISPR, they’re able to break genes in up to 75 percent of Parhyale embryos, versus a maximum 25 percent success rate with the old technique. Even better, they now have the ability to mutate several genes at once with CRISPR, which means they can now see how genes interact. When the researchers had tried to mutate multiple genes with their old technique, it rarely worked. (Though Patel notes that the older RNA technique is still very useful for certain applications).

Their research takes less time with CRISPR, too—in a study Patel’s lab published in Current Biology in 2015, they knocked out six Hox genes in about a year. Before that, they had already spent years trying to break a specific Hox gene with their old method, but were never able to do it. “Everything just goes faster,” says Patel, a professor of genetics, genomics and development. “CRISPR-Cas9 is an incredibly elegant system, and it’s very easy to control.” It also makes it simpler to study more exotic creatures (beyond the standard flies and mice), such as animals like Parhyale or butterflies. “It’s always been hard to work with a new organism,” says Jarvis, “CRISPR is awesome because suddenly, you don’t have to spend decades developing a model.” As long as you have the sequence of the gene you want to target, you’re set.

Patel’s lab is hardly the only one capitalizing on CRISPR—scientists around the world are exploring all sorts of different uses for the gene editing tool, like wiping out malaria-spreading mosquitoes, finding new ways to treat cancer, or engineering disease-resistant crops. In July, researchers announced they had successfully edited the genome of viable human embryos with CRISPR; the technique allowed them to fix a disease-causing mutation in the embryos’ DNA (though some are now skeptical of the researchers’ results). Just a few weeks later, scientists in Massachusetts reported they had made a significant advance towards pig-to-human organ transplants. They used CRISPR to inactivate 25 viruses intrinsic to pigs’ genomes, overcoming a big obstacle in making porcine transplants safe for humans.

PLAYING GOD

I had reached the end of my CRISPR experiment—so what had I learned? First, I found out it was not completely crazy for my roommate to wonder whether a DIY CRISPR kit in our fridge would make us sick. This year, German authorities restricted imports of the Odin DIY CRISPR bacteria kit after the Bavarian Health and Food Safety Authority tested two kits and found them to contain potentially pathogenic bacteria. But even the European Center for Disease Prevention and Control concluded that there was little to worry about—that “the risk of infection by the contaminating strains in the kit is low for the users … assuming that they are healthy people.” (Zayner declined to comment on the record about the incident, but he publicly posted a response on Twitter, where he criticized the methodology used by the Bavarian agency and denied wrongdoing by his company. The kits are still available for purchase online through The Odin.)

As for my bigger question—could untrained DIY-ers actually achieve scientific breakthroughs?—I asked academic researchers what they thought. Dana Carroll, for his part, believes amateurs could make meaningful discoveries. “In the professional science community, people keep coming up with new ways to use this technology—people are really only limited by their imagination,” he explains. “It’s possible that people working in their garages or their kitchens will come up with a novel application or a solution to a problem that professionals just haven’t gotten around to.” And Carroll says it would be easy for a DIY-er to share any discoveries with researchers, by attending their talks or simply by contacting them through their Web sites. Yet he notes that the DIY community faces limitations, because amateur scientists likely would lack the necessary resources. “It’s unlikely they will bring a major application all the way to fruition,” he says, “But they could certainly get started on something.”

Finally, what about the nightmare scenario: Is CRISPR so easy to use that we need to worry about biohackers—either accidentally or intentionally—creating dangerous pathogens? Carroll and others think that the danger of putting CRISPR in the hands of the average person is relatively low. “People have imagined scenarios where scientists could use CRISPR to generate a virulent pathogen, ” he says. “How big is the risk? It’s not zero, but it’s fairly small.” Gersbach agrees. “Right now, it’s difficult to imagine how it’d be dangerous in a real way,” he explains, “If you want to do harm, there are much easier and simpler ways than using this highly sophisticated genetic editing technique.”

Back in Patel’s lab, Jarvis replaced the squirming female beach hopper under my microscope with a tiny Parhyale embryo. Jarvis told me she knocked out a Hox gene called Abd-B in this one—the embryo will grow jumping legs where it should have swimming legs, and forward walking legs instead of anchor legs. At this point, it just looked like an opaque ball of goo to me.

Next to me, another grad student examined a fragment of a brown and gold butterfly wing—Patel’s lab is also knocking out butterfly genes with CRISPR to see how they build wing color. “An old grad student used to joke that we were genetically modifying the wings to make the Mona Lisa,” Jarvis told me. I laughed and glanced back under the microscope. A puff of my breath suddenly struck the Parhyale embryo. It danced wildly around the petri dish, like a grain of sand caught in a windstorm.

References
RadioLab – CRISPR
Breakthrough DNA Editor Born of Bacteria – Quanta Magazine
Rewriting the Code of Life – The New Yorker, Jan 2 2017 – Michael Spector
Emerging Technology: Concerning RNA-guided gene drives for the alteration of wild populations – Kevin M Esvelt, Andrea L Smidler, Flaminia Catteruccia, and George M Church
Wiki for CRISPR
Wiki for Gene Drive

Notes
1. For those who prefer listening to reading, RadioLab did a great podcast on CRISPR here.↩
2. UC Berkeley in California and the BROAD institute in Massachusetts are the two main adversaries and have already taken the issue of CRISPR ownership to the courts. Here is the BROAD institute’s timeline.↩
3. The implications of the Ken Thompson hack are profound. Here is Thompson’s classic essay “Reflections on Trusting Trust” and here is a good summary of the implications.↩
4. If you read about Thompson’s hack, you’ll see he actually took his hack one step further and modified the C-compiler to recognize itself and to inject the code that recognized login that injects the code in login.↩
5. This is not strictly true for all generations. Although the effectiveness should be very high at first, biology usually manages to throw in some surprises, and researchers have found that organisms actually build a sort of resistance to gene drives, much like antibiotic resistance.↩