The ability to edit RNA and individual DNA base pairs will make gene editing much more precise.
Several years ago, scientists discovered a technique known as CRISPR/Cas9, which allowed them to edit DNA more efficiently than ever before.
Since then, CRISPR science has exploded; it’s become one of the most exciting and fast-moving areas of research, transforming everything from medicine to agriculture and energy. In 2017 alone, more than 14,000 CRISPR studies were published.
But here’s the thing: CRISPR, while a major leap forward in gene editing, can still be a blunt instrument. There have been problems with CRISPR modifying unintended gene targets and making worrisome, and permanent, edits to an organism’s genome. These changes could be passed down through generations, which has raised the stakes of CRISPR experiments — and the twin specters of “designer babies” and genetic performance enhancers — particularly when it comes to editing genes in human embryos.
So while CRISPR science is advancing quickly, researchers are still very much in the throes of tweaking and refining the toolkit. And on Wednesday, researchers at Harvard and MIT launched a coordinated blitz with two big reports that move CRISPR in that safer and more precise direction.
In a paper published in Science, researchers described an entirely new CRISPR-based gene editing tool that targets RNA, DNA’s sister, allowing researchers to make transient changes to genetic material. In Nature, scientists described how a more refined type of CRISPR gene editing can alter a single bit of DNA without cutting it — increasing the tool’s precision and efficiency.
The first paper, out Wednesday in Science, describes a brand new gene editing system. This one, from researchers at MIT and Harvard, focuses on tweaking human RNA instead of DNA.
Our cells contain chromosomes made up of chemical strands called DNA, which carry genetic information. Those genes have recipes for proteins that lead to a bunch of different traits. But to carry out the instructions in any one recipe, DNA needs another type of genetic material called RNA to get involved.
RNA are ephemeral: They act like a middleman, or messengers. For a gene to become a protein, that gene has to be transcribed into RNA in the cell, and the RNA is then read to make the protein. If the DNA are permanent — the family recipe book passed down through generations — the RNA are like your aunt’s scribbled-out recipe on a Post-It note, turning up only when it’s needed and disappearing again.
With the CRISPR/Cas9 system, researchers are focused on editing DNA. (For more on how that system works, read this Vox explainer.) But the new Science paper describes a novel gene editing tool called REPAIR that’s focused on using a different enzyme, Cas13, to edit that transient genetic material, the RNA, in cells. REPAIR can target specific RNA letters, or nucleosides, that are involved in single-base changes that regularly cause disease in humans.
This is hugely appealing for one big reason: With CRISPR/Cas9, the changes to the genome, or the cell’s recipe book, are permanent. You can’t undo them. With REPAIR, since researchers can target single bits of ephemeral RNA, the changes they make are transient, even reversible. So this system could fix genetic mutations without actually touching the genome.
“With [CRISPR/Cas9] gene editing, we know we can make the desired change, but sometimes we also make off-target or unintended changes in the genome,” said the Broad Institute’s Feng Zhang, a senior author on the paper. “And whether it’s off target or unintended, it’s permanent and very difficult to reverse, which poses a safety concern.”
Because RNA is transient, once you stop editing the RNA, the edited material will get degraded over a period of time, and whatever changes were made to the cell will also disappear. “This overcomes safety concerns,” Zhang added.
“This is less scary,” said Alexis Komor, a scientist working on gene editing at the University of California San Diego who was not involved with the RNA research. “This idea of permanently changing our genome is very scary, and [REPAIR] is an alternative to that. Maybe we don’t need to permanently change everything forever. Instead, we just need a short treatment where we’re doing the modification we want for a specific period of time.”
In the paper, the researchers showed that REPAIR could be used in human kidney cells to fix the messenger RNA, leading to functional proteins. The finding builds on their earlier work on the Cas13 family of proteins: In a 2015 paper, researchers described discovering Cas13; in a 2016 paper, they described Cas13’s basic biology; earlier this year, they published papers describing how Cas13 could be used to diagnose viral or bacterial infections and how it could destroy RNA in human cells. Today — most significantly — their paper shows that you can engineer Cas13 to edit RNA in human cells.
And what’s perhaps most exciting is that the researchers who authored this study foresee clinical applications of the REPAIR system for reversing diseases in humans. The transient nature of REPAIR edits, the researchers wrote, would be useful in “treating diseases caused by temporary changes in cell state,” such as local inflammation, Type 1 diabetes, or psoriasis. If you only want to edit a protein when there’s inflammation and stop when the inflammation is gone, you can.
Researchers could also expose cells to the RNA editor during a treatment course and then stop, for example, if science advances such that a better treatment becomes available.
The new tool also has other advantages over CRISPR/Cas9. CRISPR/Cas9 relies on machinery in the cell that’s linked to cell division, so it can only be used when cells are actively replicating. If you want to fix a mutation in the brain or muscle cells — which don’t replicate — you hit major roadblocks.
“That was another reason that motivated us to develop RNA editing,” Zhang explained. “REPAIR doesn’t depend on other components in the cell, so it can modify the RNA and lead to the desired change we want” in a a broad variety of tissues, including the brain and the muscles.
“One of the things that I really care about is being able to try to treat diseases that affect the brain, and so one possibility is to use [RNA editing] to repair single-letter mutations in the genome that lead to autism, epilepsy, neurodegeneration,” he added.
The second paper, out in Nature and also by researchers at MIT and Harvard, describes a refining of CRISPR for DNA editing, this time focused on individual DNA “base pairs.”
All living organisms store genetic information in DNA, the chemical structure that serves as a recipe book for proteins. These recipes are written in an “alphabet” of just four letters: cytosine (C), guanine (G), thymine (T), and adenine (A).
The letters, or bases, form specific bonds across the double strand of DNA: C pairs with G, while T pairs with A. The human genome has 3 billion of these “base pairs” and their precise order determines not just the structure of proteins, but their quantity and the circumstances of their production — like ramping up antibodies in response to an infection.
Despite the vast library of protein recipes in the human genome, there is little margin for error.
David Liu, a chemistry and chemical biology professor at Harvard University, explained that of the more than 50,000 known genetic mutations linked to disease in humans, about 32,000 of them — illnesses like cystic fibrosis and sickle cell anemia — are caused by a mutation in a single base pair.
Fixing these mistakes requires a tool that can find a one-in-3-billion target and tweak it without introducing any new errors, an ordinarily arduous and tedious task.
In the paper, Liu and his team at the Broad Institute of MIT and Harvard reported a new technique that piggybacks on CRISPR to edit individual base pairs of DNA. The hope is that it could lead to more precise and less invasive therapies for disease.
“Standard genome editing methods, including the use of CRISPR-Cas9, make double-stranded breaks in DNA,” Liu said during a press briefing. “But when the goal is simply to fix a point mutation, base editing offers a more efficient and cleaner solution.”
He likened CRISPR-Cas9 to scissors, snipping out targeted chunks of DNA and pasting in new ones. The base editing technique his team created is more like making corrections with Wite-Out, tweaking individual base pairs in DNA in place without cutting anything out. The tool you want depends on the kinds of edits you’re making.
The most common kind of single base pair change that results in disease — accounting for half of known diseases linked to single point mutations — is the change from G-C to T-A. This happens spontaneously between 100 and 500 times per day per cell in humans, but the changes usually occur in harmless parts of DNA or are caught and fixed before they can do damage.
For the mutations that do end up slipping through and causing disease, researchers wanted a way to reverse them. However, there is no known enzyme in nature that can turn A-T pairs into G-C pairs in DNA.
Liu and his team decided to make one by evolving an enzyme called adenosine deaminase which they thought they could aim at particular genes.
To make sure the enzyme is changing the right base pair, Liu and his team attached it to a version of CRISPR-Cas9 that was modified so it can still find a specific chunk of DNA, but without making any cuts. The result is a process that is efficient and targeted, producing few unwanted changes.
The team tested the base editing process on cells encoding a mutation that causes hemochromatosis, an illness where the body stores too much iron. They found that they were able to reverse the mutation in 28 percent of cells and found no evidence of any undesired modifications. Though the modification rate was low in this initial test, this proof-of-concept highlights the potential for base editing as a precise tool to fight disease.
Liu thinks DNA base editing will be used for one-time permanent fixes to DNA, and RNA editing for temporary changes. “I’m hopeful that as complementary approaches, DNA base editing and RNA base editing will together enable an especially broad set of potential research and therapeutic applications,” he said.
Scientists are already tinkering with CRISPR to edit out diseases like HIV, reduce our reliance on petrochemicals, treat Alzheimer’s and cancer, and engineer plants to address food insecurity — and that’s just a small sampling of CRISPR’s far-reaching impact. The new approaches described today will give them even more CRISPR tools to work with.
"You’d have to be criminally reckless, or insane, to try to make a baby this way unless and until we’ve had a decade or more of preliminary research, with human tissues and with non-human animals (including certainly primates and maybe even some of the non-human apes), showing that it is safe. If the moral risk isn’t enough of a deterrent, the potential legal liability should be."
The National Institutes of Health's Francis Collins was more pointed with his concerns about the ethics of human gene editing — particularly the question of whether we are ready to make new human babies who have no say over having their DNA altered. (NIH won't fund genomic editing technologies involving human embryos.) The concept of altering the human germline in embryos for clinical purposes has been debated over many years from many different perspectives, and has been viewed almost universally as a line that should not be crossed," he wrote in a statement. (NIH won't fund genomic editing technologies involving human embryos.)
In a National Academies of Science consensus statement on gene editing science, researchers agreed that genome editing of the human germline might be permitted in the future, “but only for serious conditions under stringent oversight.”
There’s still simply a long way to go in terms of understanding basic biology.
“These are fairly sophisticated technologies and it takes a decent amount of knowhow to be able to operate them,” said Zhang. “But even more importantly, biology is very complicated. We are having a difficult time just treating diseases that are caused by a single mutation. There’s lots of biology we need to [learn] to be able to engineer biology.”
Still, that’ll change over time — and these papers represent advances in that direction.