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.