With CRISPR’s meteoric rise as a gene editing marvel, it’s easy to forget its lowly origins: it was first discovered as a quirk of the bacterial immune system.
It seems that bacteria have more to offer. This month, a team led by the famed synthetic biologist Dr. George Church at Harvard University hijacked another strange piece of bacteria biology. The result is a powerful tool that can—in theory—simultaneously edit millions of DNA sequences, with a “bar code” to keep track of changes. All without breaking a single delicate DNA strand.
For now, these biological tools, called “Retron Library Recombineering (RLR),” have only been tested in bacterial cells. But as CRISPR’s journey to gene therapy shows, even the weirdest discoveries from lowly creatures may catapult our wildest gene therapy or synthetic biology dreams into reality.
“This work helps establish a road map toward using RLR in other genetic systems, which opens up many exciting possibilities for future genetic research,” said Church.
Wait, Why Is CRISPR Inadequate?
Retrons are weird. Let’s start with CRISPR instead.
You might already be familiar with how it works. There are two components: a type of RNA, and a protein. The “bloodhound” guide RNA tethers the Cas “scissor” protein to a particular gene. In the classic version, Cas chops up the gene to turn it off. More recent advances allow Cas to replace a specific genetic letter, or snip multiple genes at once.
For the chop-and-replace version, as the gene heals itself, it’ll often seek out a template. CRISPR can carry a template gene for the cell to rely on. In this way the cell is tricked into a genetic copy edit: replacing a defective genetic sentence with one that’s biologically grammatical.
The problem with CRISPR is the chopping of DNA. If you’ve ever cut a sentence on your phone, realized you cut the wrong bits, pasted it back with another message that now doesn’t make sense, and hit send—well, that’s kind of analogous to what can happen with CRISPR. The danger of damage to our genome goes up when we need to edit multiple genes. This becomes a massive problem in synthetic biology, which uses genetic manipulation to bestow cells with new abilities, or even engineer completely new organisms.
Cells are stubborn creatures developed from eons of evolution, so changing a single gene is rarely enough to get, for example, a bacteria to pump out biofuels or medications, making multiplexed gene editing necessary. Most cells also rapidly divide, so that it’s essential for any genetic tinkering to stick across generations. CRISPR often struggles with both. The Church team thinks they have a solution.
The new tool is called RLR, and the first “R” stands for retrons. These are widespread but utterly mysterious creatures whose “natural biology…is largely unknown,” the team wrote, though similar to CRISPR, they may be involved in the bacterial immune system.
First discovered in 1984, retrons are floating ribbons of DNA in some bacteria cells that can be converted into a specific type of DNA—a single chain of DNA bases dubbed ssDNAs (yup, it’s weird). But that’s fantastic news for gene editing, because our cells’ double-stranded DNA sequences become impressionable single chains when they divide. Perfect timing for a retron bait-and-switch.
Normally, our DNA exists in double helices that are tightly wrapped into 23 bundles, called chromosomes. Each chromosome bundle comes in two copies, and when a cell divides, the copies separate to duplicate themselves. During this time, the two copies sometimes swap genes in a process called recombination. This is when retrons can sneak in, inserting their ssDNA progeny into the dividing cell instead. If they carry new tricks—say, allowing a bacteria cell to become resistant against drugs—and successfully insert themselves, then the cell’s progeny will inherit that trait.
Because of the cell’s natural machinery, retrons can infiltrate a genome without cutting it. And they can do it in millions of dividing cells at the same time.
“We figured that retrons should give us the ability to produce ssDNA within the cells we want to edit rather than trying to force them into the cell from the outside, and without damaging the native DNA, which were both very compelling qualities,” said study author Dr. Daniel Goodman.
The Making of RTR
Similar to CRISPR, RTR has multiple components: the genetic snippet that contains a mutation (the bait), and two proteins, RT and SSAP (reverse transcriptase and single-stranded annealing proteins), that transform the retron into ssDNA and let it insert itself into a dividing cell.
Still with me?
Like Game of Thrones, there’s a lot of players. So to make it clearer: retrons carry the genetic code we want to insert; RT makes it into a more compatible form that’s called ssDNA; and SSAP sticks it into DNA as it’s dividing. Basically, a Trojan horse invades the cell and pours out spies that insert themselves into the cell—changing its DNA—with the help of enzymatic magicians.
The two proteins are new to the party. Previously, scientists have tried to use retrons for gene editing, but the efficiency was extremely low—around 0.1 percent of all bacterial cells infected. The two newcomers quieted down the bacteria’s natural “alarm system” that corrects DNA changes—so they ignore the new DNA bits—and allow edits to enter and pass on to the next generation. One other trick was to neuter two genes that encode for proteins that normally destroy ssDNA.
In one test, the team found that over 90 percent of bacterial cells readily admitted the new retron sequence into their DNA. They next went big. Compared to CRISPR, retrons have a leg up in that their sequence can act as a bar code. This means it’s possible to perform multiple gene editing experiments at once, and figure out which cells were edited with what retron by sequencing the bar code.
In a proof-of-concept test, the team blasted some bacteria cells with retrons that contained sequences for antibiotic resistance. By sequencing the retron DNA letters alone from a pool of bacteria treated with antibiotics, they found that cells with retrons—giving them the new superpower against drugs—remained in far higher portions than other cells.
In another test, the team tried to determine how many retrons they could use at once. They took another strain of bacteria that’s resistant to antibacterials, and chopped up its genome to build a library of tens of millions of retrons. They then stuck these chunks into hula hoops of DNA—called plasmids—and floated them into bacteria cells. As before, the team could easily find the retrons that conferred anti-bacterial power by sequencing the bar codes of those that remained alive.
That’s the how. But what’s the why?
The goal is easy: to find another solution to CRISPR that can influence millions of cells at once, without damaging the cells. In other words, take gene editing into the big data era, through multiple generations.
Compared to CRISPR, the new RLR tool is simpler because it does not require a “guide” tool in addition to an “editing” tool—a retron is basically a two-in-one. Being able to influence multiple genes at once—without physically cutting into them—also makes it an intriguing tool for synthetic biology. The tool’s also got staying power. Instead of a “one and done” CRISPR ethos, it lasts through generations as cells divide.
That said, RTR’s got competition. Because it works best with dividing cells, it might not be as powerful in reluctant cells that refuse to split—for example, neurons. For another, recent upgrades to CRISPR have made it possible to also turn genes on or off—without cutting them—through epigenetics.
But RLR offers scale. “Being able to analyze pooled, barcoded mutant libraries with RLR enables millions of experiments to be performed simultaneously, allowing us to observe the effects of mutations across the genome, as well as how those mutations might interact with each other,” said Church.