Editing Humanity: The CRISPR Revolution and the New Era of Genome Editing

CHAPTER 2 A CUT ABOVE

On June 26, 2000, President Bill Clinton walked into the East Room of the White House flanked by two famous scientists, Francis Collins and Craig Venter. Clinton announced a landmark in the Human Genome Project (HGP)—the first rough draft of the human genome sequence, the book of life. Beaming in via satellite from 10 Downing Street was British prime minister Tony Blair, waving the flag for the British team that chipped in about a third of the sequence.

For two years, two teams—small armies more like—of scientists had been dueling to reach this epic milestone. In one corner was Collins, the field marshal of the international government-funded alliance to decode human DNA. The prize was a treasure map of the human genome, displaying the order of 3 billion letters of the DNA alphabet (a four-letter code of chemicals abbreviated by A, C, T, and G) bundled into twenty-three pairs of chromosomes.

In the opposite corner was Venter, a maverick scientist/entrepreneur who brazenly launched a hostile takeover of the genome project. He divulged his plans to Collins in the United Airlines lounge at Washington Dulles airport, and then to the world via the front page of the New York Times. His new company, Celera Genomics, vowed to cut through years of government inefficiency and bureaucracy to compile the sequence faster and cheaper using a warehouse full of the latest automated sequencing machines—each named after a sci-fi character—plus a massive Compaq supercomputer to crunch the data. As a consolation prize, Venter said Collins could sequence the mouse genome instead. Almost overnight, the tables had been turned: the “Darth Vader” of genomics had the weaponry and momentum; Collins and his allies were the plucky overmatched rebels with their backs against the wall.

As public bickering between the factions degenerated into outright hostility, it threatened to tarnish the reputations of the project leaders, not to mention the purpose of the mission. The White House helped orchestrate a temporary ceasefire to facilitate a historic celebration.¹ Clinton hailed the achievement as “the most important, most wondrous map ever produced by humankind… The language in which God created life.” GENETIC CODE OF HUMAN LIFE IS CRACKED BY SCIENTISTS was the banner headline on the front page of the New York Times.²

But who was the owner of said cracked code? The NIH consortium had collected DNA from dozens of anonymous volunteers who answered a March 1997 newspaper ad placed in the Buffalo News by molecular geneticist Pieter de Jong (the Master Chef of building DNA libraries). Years later, genetic analysis revealed that the largest single contributor, code-named RP11, was likely to be African American.³ Like everyone else, RP11 and the other DNA donors were mutants, each carrying hundreds or thousands of DNA variants predisposing to rare and common diseases, including type 1 diabetes and hypertension.⁴ Celera had selected DNA from five volunteers of diverse ethnic backgrounds; Venter later admitted he was one of the chosen few.

Reading the book of life—even if at this stage there were many pages missing or torn or out of order—was a monumental achievement. This was the moonshot of biology, arguably the biggest event since Crick and Watson assembled the double helix in 1953. We had become the first species to translate the instruction manual, even if we couldn’t describe how much of it works. Textbook chapters proclaiming that humans possess more than 100,000 genes were rendered obsolete as we were humbled to learn that our genome contains barely 20,000.

One of the biggest champions of the HGP was Sir John Maddox, the editor emeritus of Nature. In 1999, Maddox published an ambitious book few would dare undertake, entitled What Remains to Be Discovered. Maddox wrote:

It is likely that the deeper knowledge of the working of the human genome now being won will suggest ways in which the design of Homo sapiens provided by 4.5 million years of natural selection could be decisively improved upon by genetic manipulation. After all, people are now manipulating the genetic structure of genes so as to make plants resistant to infections. Why not manipulate the human genome to the same end? It is a reasonable guess that Homo sapiens will not always disclaim such opportunities.⁵

As he wrote those words, a band of scientists worlds away from the television cameras and presidential plaudits were taking the first steps toward developing a new technology that could tinker with the code we had just spent some $2 billion over a decade to spell out. It was the dawn of genome editing.

Editing is an essential step in creating works of literature, or music, or art. The fortunes of many blockbuster films might have been very different if producers had gone with their original titles. Alien was going to be called “Star Beast,” Back to the Future was almost released as “Spaceman from Pluto,” and the working title of Pretty Woman was “3,000.” Jane Austen’s “First Impressions” became Pride and Prejudice. Margaret Mitchell’s Scarlett O’Hara was originally named Pansy. “Editing, of text literary or genetic, (almost) always makes things better,” writes Fyodor Urnov.⁶

While I was watching the rapid progress in high-throughput DNA sequencing in the 2000s, scientists were devising a molecular word processor to edit the book of life—to search, cut, and paste words and letters, identifying typos, deleting misspellings, and pasting in corrections. Within a decade of crowning ourselves the first species to decode our genetic script, we were already testing our ability to engineer changes in any organism on a whim. Taken to its logical conclusion, we can now redirect and accelerate our own evolution, and that of almost every organism on earth.

“This is the nature of discovery,” says geneticist Shirley Tilghman, former president of Princeton University. Every major scientific discovery has the capacity to be deployed for good and ill. “It’s going to take wise societies to direct those discoveries down the right path.”⁷ The rapid development of genome editing is a daunting, unprecedented, and in some ways frightening responsibility. One that has already been violated.

Before we go any further, let’s consider what is so special about this revolutionary technology with the funny name that sounds like a cross between a candy bar and a refrigerator drawer. In striving to paint a picture of CRISPR, writers have reached for one metaphor after another: the hand of God, a bomb disposal squad, a pencil eraser, a surgeon’s scalpel, a retinal scanner, and frequently, a “molecular scissors.”⁸ STAT produced a top ten list of CRISPR analogies, culminating in the Offiziersmesser, better known as the Swiss Army knife of molecular biology. Likewise, CRISPR is more than just a single sharp blade for cutting DNA, but an ever-expanding array of molecular gadgets for editing and manipulating DNA with ever greater finesse and flexibility.

CRISPR is one of those once-in-a-generation breakthroughs that changes the way science is conducted almost overnight. Ironically, the technology harnessed from a bacterial antiviral immune system went viral. But it was not the first technique for genome editing. Earlier methods for gene editing were conceived in the early 2000s, refined, and even entered the clinic before the advent of CRISPR. Urnov and his colleagues at Sangamo coined the term “genome editing” in 2005 while refining a technology called zinc finger nucleases (ZFNs), which is still in clinical use. In 2011, the year before CRISPR burst into the scientific mainstream, the journal Nature Methods anointed genome editing its “Method of the Year.” ZFNs and another gene-editing platform called TALENs have their admirers, but were too fussy and expensive to break out the way CRISPR has.

CRISPR takes the premise of other forms of genome editing and (in the parlance of Spinal Tap) turns it up to 11. From Australia to Zaire, researchers worldwide are using CRISPR to edit genes in almost any organism on planet earth. The ease of uptake stems from the fact that CRISPR is, in essence, a technology honed by evolution over hundreds of millions of years. CRISPR doesn’t require expensive lab instruments such as $1-million state-of-the-art DNA sequencing machines—most of the reagents can be ordered over the Internet and handled in the lab without any special safety precautions, just as Zhang demonstrated for 60 Minutes. High-school students can learn the fundamentals of CRISPR in a biology classroom.⁹ A nonprofit in Boston called Addgene serves as a clearing house for CRISPR reagents. By early 2020, Addgene had distributed more than 180,000 CRISPR constructs to more than 4,000 laboratories around the world, according to director Joanne Kamens.¹⁰

In the summer of 2012, the groups of Charpentier and Doudna demonstrated that they could take the bacterial CRISPR system and, with some nifty molecular tweaking, transform it into an exquisitely tunable genetic cursor that could be used to cut more or less any specific stretch of DNA. Rodolphe Barrangou, the chief editor of The CRISPR Journal, calls that study a tipping point that showed that “you could repurpose this cool, idiosyncratic, revolutionary immune system in bacteria and turn that into a tool that people can use readily in the lab to cut DNA.”¹¹ Six months later, Zhang’s group, in collaboration with the Rockefeller University’s Luciano Marraffini, and independently George Church’s group, demonstrated that the CRISPR-Cas9 tool could effectively edit mammalian DNA. “That changed the world,” says Barrangou.

Indeed, around the world researchers seized this simple, programmable gene-editing tool, producing new discoveries that flew into the pages of the top science and medical journals. Stanford law professor Hank Greely offers a nice analogy. “The Model T was cheap and reliable, and before long everybody had a car and the world changed. CRISPR has made gene editing cheap, easy and accessible… I think it’s going to change the world,” he says. “Exactly how beats me.”¹²

The incandescent rivalry between the two giants of soccer in Buenos Aires—River Plate and Boca Juniors—has been called eternal. But there is a rivalry that has shaped life on earth from the beginning and rages all around us to the present day. The most important arms race on the planet takes place between two implacable enemies, the nuclear superpowers of the microbial world—bacteria and the viruses (or bacteriophages) intent on their mutual destruction. This war has raged for life eternal, a billion years at least.

We didn’t need to experience the COVID-19 pandemic to know that viruses are the invisible menace, harbingers of sickness and death. “The single biggest threat to man’s continued dominance on this planet is the virus,” Nobel laureate Joshua Lederberg famously said. Beyond social distancing and some natural immunity, the human species mounts a variety of countermeasures, including vaccines and a battery of tailored or repurposed drugs and therapies. The threat is never extinguished, because viruses are able to mutate, evolve, capture genetic material from their hosts, and continually reinvent themselves.

Bacteria know how we feel. They face a constant viral threat of their own from bacteriophages—viruses that exclusively infect bacteria. There are an unfathomable 10 nonillion (10³¹) phages on planet earth—one trillion for every grain of sand.^I “Don’t ask me how people calculate this number, but I believe them,” says Marraffini.¹³ Laid end to end, those submicroscopic phages would stretch 200 million light-years.¹⁴ Under the electron microscope, many look quite menacing, like a cross between the lunar lander and a spider, legs splayed to hook onto the cell surface; others have the innocent charm of a circle lollipop with a long tail. Once attached, the virus impregnates the bacterium with its own genetic material, a short strand of either DNA or its chemical cousin RNA, hijacking the host’s protein-manufacturing machinery. Within twenty to thirty minutes, scores of freshly assembled viral progeny burst out of the now defunct host cell like a hundred Aliens erupting out of John Hurt’s stomach. “The cells explode, they pop,” like a balloon, says Marraffini.

Surrounded by would-be phage invaders, bacteria have evolved a variety of defense systems to surveil and destroy this threat. When I was studying biochemistry in the 1980s, we learned that bacteria boast an army of potent enzymes that recognize and attack specific motifs in any foreign DNA. (The same sequences in the bacterial DNA are protected from those same nucleases with chemical tags, like a child-safety electrical outlet cover.) Scientists seized on these restriction enzymes as a means to cut, swap, and ligate DNA fragments, for example pasting human genes into bacteria, giving birth to the biotechnology industry. But as we shall see later, we now know that bacteria possess another immune system. CRISPR is a small subsection of the bacterial genome that stores snippets of captured viral code for future reference, each viral fragment (or spacer) neatly separated by an identical repetitive DNA sequence. Think of it as an FBI filing cabinet of Most Wanted offenders.

CRISPR is more than just a vault of viral villainy; within reach is the armory for a potent ground-to-air missile defense system. When the cell detects an invading virus, the first step is to activate the CRISPR array, producing an RNA copy of the archived viral sequences. This RNA string is then sliced up into individual sequences, each fragment derived from a different virus and serving as a police artist’s sketch of a possible offender. The RNA can’t do any damage by itself, so it is weaponized by binding to a DNA-cutting enzyme called Cas (CRISPR-associated sequence), forming a ribonucleoprotein complex that is armed with a GPS signal and ready to do battle.

Phages and the CRISPR Pathway. (A) Caught in the Act: Phages land on the surface of E. coli to launch their attack. (B) CRISPR–Cas immunity. 1. Bacteria capture fragments of viral DNA and integrate these spacers into the expanding CRISPR array. 2. To combat a phage infection, the CRISPR array (pre-crRNA) is transcribed into RNA, then processed into mature crRNAs. 3. In the interference stage, the crRNA and Cas protein(s) form a complex that targets the corresponding phage sequences for degradation. Some CRISPR systems (Class 1) feature multiple Cas proteins as shown, whereas the simpler Class 2 systems require only a single nuclease such as Cas9. (Adapted from ref. 15.)

There are half-a-dozen different flavors or types of CRISPR system in the microbial universe, which are organized into two classes based on their architecture and other properties.¹⁵ One of the simplest arrangements—Type II—features an enzyme called Cas9. This nuclease makes a clean break on both strands of the DNA double helix like a pair of nail clippers, but not indiscriminately. It grabs an RNA tag, holding it like a mugshot, searching the incoming DNA for a match. Once encountered, Cas9 will latch onto the viral DNA and cut it, neutralizing the threat. Cas9 is “truly wondrous,” Urnov explains. “When Cas9 polices the intracellular neighborhood for invasions, it literally carries a copy of that most wanted poster with it. Asking everyone that comes in: “Excuse me, do you carry an exact match to this little most wanted poster that I’m carrying? Yes? Then I’ll cut you.”¹⁶

Classes of CRISPR. There are several flavors of CRISPR, which are categorized into two broad classes, 1 and 2. In Class 1, the DNA cleavage is performed by a complex of proteins, sometimes called Cascade. In Class 2, CRISPR systems feature a single Cas nuclease such as Cas9, Cas12 or Cas13. (For details, see ref. 15).

Marraffini reveals how the two bacterial defense systems complement each other. Restriction enzymes offer the first barrier of defense against the viral menace, shredding the viral DNA into pieces that can be incorporated into the CRISPR array. But if phages, ever evolving, dodge the first line of defense, CRISPR immunization kicks in. It is analogous to vaccination, Marraffini says. “When the phage DNA is dead, CRISPR can scavenge spacers to immunize the host.” Only a very few infected bacteria actually acquire spacers—about 1 in 10 million—but that provides one cell with the power to vanquish the viral threat and rebuild the population.^II

In Adam Bolt’s 2019 documentary Human Nature, we meet David Sanchez, a charming boy who suffers from sickle-cell disease. As he learns about the potential of CRISPR to cure his disease, he asks perceptively: “How does this thing work and know how to target the right gene, not the gene that makes hair?”

The genius of the CRISPR revolution was to parcel Cas9 not with a virally derived RNA, as in nature, but with a synthetic guide RNA programmed by researchers that allows them to target more or less any DNA sequence in any gene in any organism. The result is we have hijacked a bacterial enzyme a billion years old and repurposed it into a 21st-century molecular scalpel for precision gene surgery. Whether we want to edit the genome of a hamster or a human, a mosquito or a mouse, a redcurrant or a redwood, the process essentially is the same. That’s because all organisms in nature use the same inert DNA code, composed of the same four-letter alphabet.

In its natural state, Cas9 is rather disinterested in DNA, essentially colliding randomly and bouncing off. But once Cas9, which has a hand-shaped structure, clasps a guide RNA, a subtle reconfiguration of the protein’s structure primes it to react with DNA as it goes in search of its matching target. According to Blake Wiedenheft, a professor at Montana State University, the Cas protein complexes “patrol the entire intracellular environment, find and bind this foreign [viral] DNA, and mark that foreign DNA for destruction in a matter of minutes… that’s a pretty remarkable task.”¹⁷

The task of finding and binding the target sequence is a two-step process. First, Cas9 seeks out and interacts with a short motif in the DNA called the PAM^III sequence—a beacon that provides the enzyme with a cue to briefly caress the DNA. “That ephemeral interaction results in a distortion of the DNA,” explains Wiedenheft. By bending the DNA, Cas9 unzips the double helix to allow the guide RNA to slip into the resulting crevice (forming a so-called R-loop).¹⁸ The guide conducts a quick sequence check against the target DNA. If a perfect match is found along all twenty or so bases, this marks the DNA sequence for destruction. Cas9 severs^IV both strands of the DNA as cleanly as a kitchen knife, creating a double-strand break (DSB) just a few bases away from the PAM sequence.¹⁹

This remarkable process was captured in a stunning video shot by University of Tokyo researchers Hiroshi Nishimasu and Osamu Nureki in 2017. Using a technique called high-speed atomic force microscopy, they were able to zoom in at the precise moment that Cas9 grasps the DNA. In the film, Cas9 looks like a gold-colored rock as it pauses over a strand of DNA for several seconds before guillotining the DNA in half.²⁰ The clip went viral after Nishimasu posted it on his Twitter account and it was shown on Japanese television.

But repurposing Cas9 to seek out a specific unique sequence in the human genome is literally a million times more complicated than cutting viral DNA. As the Cas9 complex enters the alien surroundings of a cell nucleus, it is confronted by a maze of DNA—twenty-three pairs of chromosomes, six billion letters of DNA—compared to a typical phage genome of just a few thousand bases. Once in the nucleus, each Cas9 molecule scours the densely packed coils of DNA to identify PAM sites, which occur on average once every full 360° rotation of the double helix. In principle, the enzyme has to interrogate 300–400 million bases to identify its precise target.

Johan Elf, a biophysicist at Uppsala University in Sweden, calculates that Cas9 normally takes about six hours to search through every PAM sequence in the bacterial genome, pausing at each prospective site for a mere twenty milliseconds to peer into the double helix to see if it has found the correct target.²¹ But the packaging of DNA in a eukaryotic cell nucleus is far more complex than bacteria. During lectures to his students at the University of Edinburgh, Andrew Wood shows a diagram of a bacterial cell alongside a winding, looping mammalian DNA fiber. “Cas9 didn’t evolve to work in the environment in which we now put it,” he says. “It’s mind-boggling that it is possible to interrogate hundreds of millions of nucleotides in a matter of hours.”²²

Once Cas9 has cut the DNA, the cell’s DNA repair enzymes reseal the break. Experts marvel that it works as well as it does.²³ Cas9 even surpasses the previously developed ZFN and TALEN^V gene-editing platforms. “They both evolved to regulate eukaryotic DNA and yet Cas9 seems to outperform them,” Wood says.

Let’s pause to note that the PAM sequence has a critical role: by searching for a short PAM sequence rather than having to unzip and check essentially the entire genome, the task of Cas9 to latch onto its target sequence is greatly simplified. The PAM also answers the riddle of how Cas9 doesn’t accidentally carve up the repeats in the CRISPR array. That’s because when they are initially added to the bacterial CRISPR array, the PAM sequence is clipped off. Genome engineers refuse to be limited by the natural list of PAM sequences, so they are modifying the original Cas9 and Cas enzymes from other species to expand their PAM preferences.

With such an effective security system, one might reasonably ask: why aren’t all viruses extinct? Viruses have sneakily evolved a multitude of escape mechanisms—a group of proteins that are able to disable the Cas nucleases, known as anti-CRISPR proteins. Bacteria and their viruses are like prey and predators locked in a perpetual battle that rages on after hundreds of millions of years.²⁴ CRISPR is found in 40 percent of bacterial genomes, and almost all archaeal genomes, but surprisingly not at all in the genomes of higher organisms. Although Cas9 is by far the most popular enzyme used in CRISPR applications—and subject to a bitter patent dispute I’ll discuss later—this enzyme represents a blip in the diverse CRISPR systems seen in nature. A huge effort is underway to mine the biological diversity on earth to uncover new Cas family proteins with novel functions to expand the CRISPR toolbox.²⁵

Once a researcher has identified the gene sequence they wish to target, they can go to any number of websites, key in the desired matching sequence, and order that custom short guide RNA sequence. If CRISPR is a molecular word processor, then the RNA acts as the “CTRL-F” function, targeting the gene sequence of interest. Cas9 acts as the “CTRL-X” keystroke. But genome editing isn’t just about pointing the cursor to highlight and remove a typo. It’s about deciding and managing what happens next—how to correct the typo.

CRISPR Cutting of DNA. 1. Scanning: The Cas9 nuclease is bound to a guide RNA in a ribonucleoprotein complex. The guide consists of CRISPR RNA (crRNA) and the tracrRNA. The Cas9 complex scans the DNA in search of a PAM sequence, which is the cue to check for a sequence match. 2. Locking In: Cas9 binds to the DNA and unzips the double helix, allowing the crRNA to align to the single-stranded DNA. 3. Cutting: If there is a perfect DNA:RNA match, Cas9 undergoes a conformational change resulting in both DNA strands being cut in the same position. (Adapted from ref. 23.)

Cells possess multiple molecular pathways to repair breaks and other mutations in DNA; if they didn’t, we wouldn’t be alive. The two most common repair pathways are called non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ sloppily stitches the broken ends of DNA back together, but frequently results in small insertions or deletions at the repair site. This is ideal for investigators using CRISPR to deliberately disrupt the function of a gene by breaking it and introducing various random insertions and deletions. The other pathway, HDR, makes a faithful repair if a suitable template is available. In normal circumstances, the template is the corresponding gene on the sister chromosome. The beauty of the CRISPR genome editing is that the investigator can supply a suitable template containing the desired sequence to patch into the Cas9-induced break, thereby resulting in the desired edit.^{VI 26}

In January 2020, about five hundred scientists flocked to Banff, a ski resort in the Canadian Rockies, for the first big CRISPR conference of the year. (It also turned out to be the last, as the COVID-19 pandemic shut down all conference travel.) The organizers invited Doudna to deliver the opening keynote address on a Sunday morning, a role to which she has grown accustomed. She began with a heartfelt apology for not being able to stay and mingle for the next several days, but she had to get back to Berkeley to give a Monday morning lecture to six hundred undergraduates.

Doudna’s lecture, delivered with the same humility and wonder at the march of science that she had at the start of her career more than three decades ago, was the biotech equivalent of a State of the Union address. Her opening summation—a tribute not only to her own work but also that of legions of researchers over the previous quarter century—was simple:

“Precision editing of any genome is within reach.”²⁷

I. The late Roger Hendrix, a renowned microbiologist, came up with the estimate of 10³¹ phage (10,000,000,000,000,000,000,000,000,000,000) on the planet, making them the most prevalent biological entity.

II. In 2020, Rotem Sorek’s group at the Weizmann Institute of Science in Israel reported a new bacterial back-up anti-phage defense system called retrons.

III. PAM stands for Protospacer Adjacent Motif. Different Cas enzymes recognize different PAMs, ranging from three to six bases. The most commonly used Cas9, from Streptococcus pyogenes, recognizes a triplet sequence, NGG, where N can be any of the four bases.

IV. Cas9 actually has two active sites, providing two separate cutting actions, one for each strand of the double helix.

V. ZFN, zinc finger nuclease; TALEN, transcription activator-like effector nuclease (see chapter 8).

VI. It will never catch on, but Patrick Harrison, a geneticist at Trinity College, Dublin, came up with a modified definition of CRISPR, a mnemonic that explains the editing/repair process: Cut—Resect—Invade—Synthesis—Proofread—Repair. On Last Week Tonight, comedian John Oliver had his own irreverent definition: Crunchy-Rectums-In-Sassy-Pink-Ray-Bans.