New tools can fix genes one letter at a time | Science News for Students

New tools can fix genes one letter at a time

These 'editors' can fix DNA mistakes that may underlie disease
Dec 19, 2017 — 6:45 am EST
DNA eraser
Scientists have used two new versions of CRISPR technology to edit DNA one letter at a time. The new tools change bits of DNA like a pencil, instead of cutting it like scissors.
Wildpixel/iStockphoto

There’s a fairly simple alphabet that spells out the directions for what genes want the body’s cells to do. But sometimes there’s a mistake — essentially a typo — in those directions. Now, working somewhat like a pencil, new tools can edit the body’s genes one letter at a time. They can fix about half of the simple typos that cause human diseases.

The new tools are based on a technology called CRISPR/Cas9. It’s like a set of molecular scissors that snips DNA. Scientists can guide the scissors to the exact spot in an organism’s genetic instruction book where they want to cut something out. They’ve used the tool to make faulty genes — mutations — or correct them. And they’ve done this in animals and in human cells.

Scientists also have ways to use CRISPR/Cas9 to change gene instructions without cutting DNA at all. These tools are called “base editors.” A DNA base is a molecule represented by a single letter in the body’s genetic code. There are four DNA bases, known as A, C, T and G.

Base editors can fix one-letter typos in the genetic code. So far, they’ve only been able to fix one type of error: They’ve turned C’s into T’s.

Using these tools, scientists have changed genes in plants, fish, mice — even in human embryos.

Editing DNA in this way may be safer than cutting it, says Gene Yeo. He’s a biologist at the University of California, San Diego. “We know there are drawbacks to cutting DNA,” he says. After CRISPR breaks DNA, a cell’s own machinery pastes it back together. But mistakes often occur during this process. And CRISPR sometimes makes extra DNA cuts at places similar to the target. That means it can create new mutations by accident. Such “permanent irreversible edits at the wrong place in the DNA could be bad,” Yeo says.

Two papers now describe different ways to solve that problem.

David Liu and colleagues tweaked the CRISPR/Cas9 gene editor so that it turns the DNA base adenine (A) into guanine (G). They shared their work October 25 in Nature.

In a second study, Feng Zhang and colleagues redesiged a gene editor called CRISPR/Cas13 to correct the same typos in RNA. RNA is a molecule similar to DNA. RNA carries messages inside cells. This study was also published October 25, but in the journal Science.

The new tools expand the toolkit scientists have for correcting diseases. Now researchers can rewrite all four letters in the genetic code.

Repairing rungs

DNA has two strands that give it the shaped of a long, twisted ladder. Bases on one side of the ladder always pair up with certain bases on the opposite side. The molecule adenine (A) always pairs with thymine (T). Guanine (G) always pairs with cytosine (C). These base pairs form the ladder’s rungs.

base pair changes
The new base editors start with a “dead” version of CRISPR/Cas9 (one that doesn’t cut DNA). Attached to that are enzymes that can change the chemical structure of DNA bases. In 2016, researchers developed an editor that changes C-G base pairs to T-A pairs (top). A newer editor (bottom) uses a different enzyme to change A-T base pairs to G-C pairs.
E. Otwell

Mutations that change C-G base pairs to T-A pairs happen all the time. Some 100 to 500 of these changes occur every day in everyone’s body. Most of those mutations are likely harmless. But some can cause serious changes to a gene’s instructions — changes that lead to disease.

Liu is a biological chemist at Harvard University in Cambridge, Mass. Scientists have linked some 32,000 DNA mutations to genetic diseases in people, he notes. And about half are the C-G to T-A swaps. Until now, he says, there was little anyone could do about them.

RNA is the chemical cousin of DNA. It uses nearly the same code to spell out genetic information as DNA does. But instead of the base thymine (T), RNA uses uracil (U). In RNA, some naturally occurring enzymes can reverse the mutations that turn C-G pairs into T-A pairs. These enzymes chemically turn adenine (A) into another molecule called inosine (I). The cell reads this I as a G. Such RNA editing happens frequently, and naturally, in octopuses and other cephalopods. Sometimes it also happens in people.

Zhang and colleagues made one of these RNA-editing enzymes into a programmable gene-editing tool. Zhang is a scientist at the Broad Institute of MIT and Harvard in Cambridge, Mass. He also was one of the first developers of CRISPR.

To create their new tool, these researchers started with CRISPR/Cas13. Normally, this tool works like a molecular scissors that cuts RNA. The researchers tweaked the tool so that it grasped RNA instead of slicing it. Then they attached the part of the RNA enzyme that converts A to I.

The researchers called the new creation REPAIR. They tested it on human cells growing in dishes. The tool edited up to about 27 percent of the RNAs of two genes. The researchers did not find any unwanted changes.

Permanent fixes

Editing RNA is good for temporary fixes, such as turning off proteins that cause inflammation. But many mutations need permanent DNA fixes, notes Liu.

Last year, his team made a base editor that converts C to T. Other researchers have used it to fix a blood disease in human embryos. But that tool couldn’t make the opposite change, switching an A to a G.

Unlike with RNA, there’s no enzyme that naturally changes A’s to G’s in DNA. So Nicole Gaudelli forced E. coli bacteria to evolve one. She works in Liu’s lab.

First Gaudelli engineered bacteria to have a mutation in a gene that helps them fight an antibiotic. Now when she fed the bacteria that antibiotic, it killed them. Only by correcting the mutation, switching an A to G, would the bacteria live. It took lots of work, but eventually the bacteria succeeded.

The researchers called this new DNA-base converter “TadA.” Gaudelli attached it to a “dead” version of Cas9, one that couldn’t cut both strands of DNA. The result was a base editor that could switch A-T base pairs into G-C pairs. The scientists named their new tool ABE. In tests on human cells, it worked in about half of all cells tested.

This base editor works more like a pencil than scissors, says Liu. In lab dishes of human cells, his team corrected a mutation. The cells came from a patient with a disorder that affects how the blood stores iron. In other cells, the team re-created helpful mutations that protect against a disease called sickle cell anemia.

Such base editing seems safer than traditional cut-and-paste editing, other researchers now report in the October issue of Protein & Cell.

Liu’s results seem to agree. His team found that cut-and-paste CRISPR/Cas9 edits made changes at nine of 12 possible “off-target” sites. This happened about 14 percent of the time. But the new A-to-G base editor changed just four of the 12 off-target sites. And those errors happened only 1.3 percent of the time.

That’s not to say cut-and-paste editing isn’t useful, Liu says. “Sometimes, if your task is to cut something, you’re not going to do that with a pencil. You need scissors.”

Editor’s note: Feng Zhang is on the board of trustees of the Society for Science & the Public, which publishes Science News for Students.

Power Words

(for more about Power Words, click here)

anemia     A disease caused by not having enough red blood cells. This reduces the body’s ability to carry oxygen efficiently to all tissues.

antibiotic     A germ-killing substance, usually prescribed as a medicine (or sometimes as a feed additive to promote the growth of livestock). It does not work against viruses.

bacteria     (singular: bacterium) Single-celled organisms. These dwell nearly everywhere on Earth, from the bottom of the sea to inside other living organisms (such as plants and animals).

base    (in genetics) A shortened version of the term nucleobase. These bases are building blocks of DNA and RNA molecules.

base pairs     (in genetics) Sets of nucleotides that match up with each other on DNA or RNA. For DNA, adenine (A) matches up with thymine (T), and cytosine (C) matches up with guanine (G).

biology     The study of living things. The scientists who study them are known as biologists.

Cas9     An enzyme that geneticists are now using to help edit genes. It can cut through DNA, allowing it to fix broken genes, splice in new ones or disable certain genes. Cas9 is shepherded to the place it is supposed to make cuts by CRISPRs, a type of genetic guides. The Cas9 enzyme came from bacteria. When viruses invade a bacterium, this enzyme can chop up the germs DNA, making it harmless.

cell     The smallest structural and functional unit of an organism. Typically too small to see with the unaided eye, it consists of a watery fluid surrounded by a membrane or wall.

cephalopods     Ocean-dwelling animals that include squid and octopuses.

chemical     A substance formed from two or more atoms that unite (bond) in a fixed proportion and structure. For example, water is a chemical made when two hydrogen atoms bond to one oxygen atom. Its chemical formula is H2O. Chemical also can be an adjective to describe properties of materials that are the result of various reactions between different compounds.

colleague     Someone who works with another; a co-worker or team member.

CRISPR     An abbreviation — pronounced crisper — for the term “clustered regularly interspaced short palindromic repeats.” These are pieces of RNA, an information-carrying molecule. They are copied from the genetic material of viruses that infect bacteria. When a bacterium encounters a virus that it was previously exposed to, it produces an RNA copy of the CRISPR that contains that virus’ genetic information. The RNA then guides an enzyme, called Cas9, to cut up the virus and make it harmless. Scientists are now building their own versions of CRISPR RNAs. These lab-made RNAs guide the enzyme to cut specific genes in other organisms. Scientists use them, like a genetic scissors, to edit — or alter — specific genes so that they can then study how the gene works, repair damage to broken genes, insert new genes or disable harmful ones.

disorder     (in medicine) A condition where the body does not work appropriately, leading to what might be viewed as an illness. This term can sometimes be used interchangeably with disease.

DNA     (short for deoxyribonucleic acid) A long, double-stranded and spiral-shaped molecule inside most living cells that carries genetic instructions. It is built on a backbone of phosphorus, oxygen, and carbon atoms. In all living things, from plants and animals to microbes, these instructions tell cells which molecules to make.

E. coli     (short for Escherichia coli) A common bacterium that researchers often harness to study genetics. Some naturally occurring strains of this microbe cause disease, but many others do not.

embryo     The early stages of a developing organism, or animal with a backbone, consisting only one or a few cells. As an adjective, the term would be embryonic — and could be used to refer to the early stages or life of a system or technology.

enzymes     Molecules made by living things to speed up chemical reactions.

evolve     (adj. evolving) To change gradually over generations, or a long period of time. In living organisms, such an evolution usually involves random changes to genes that will then be passed along to an individual’s offspring. These can lead to new traits, such as altered coloration, new susceptibility to disease or protection from it, or different shaped features (such as legs, antennae, toes or internal organs). Nonliving things may also be described as evolving if they change over time. For instance, the miniaturization of computers is sometimes described as these devices evolving to smaller, more complex devices.

gene     (adj. genetic) A segment of DNA that codes, or holds instructions, for a cell’s production of a protein. Offspring inherit genes from their parents. Genes influence how an organism looks and behaves.

genetic     Having to do with chromosomes, DNA and the genes contained within DNA. The field of science dealing with these biological instructions is known as genetics. People who work in this field are geneticists.

guanine     One of four substances that organisms need to produce DNA.

inflammation     (adj. inflammatory) The body’s response to cellular injury and obesity; it often involves swelling, redness, heat and pain. It also is an underlying feature responsible for the development and aggravation of many diseases, especially heart disease and diabetes.

iron     A metallic element that is common within minerals in Earth’s crust and in its hot core. This metal also is found in cosmic dust and in many meteorites.

molecule     An electrically neutral group of atoms that represents the smallest possible amount of a chemical compound. Molecules can be made of single types of atoms or of different types. For example, the oxygen in the air is made of two oxygen atoms (O2), but water is made of two hydrogen atoms and one oxygen atom (H2O).

mutation     (v. mutate) Some change that occurs to a gene in an organism’s DNA. Some mutations occur naturally. Others can be triggered by outside factors, such as pollution, radiation, medicines or something in the diet. A gene with this change is referred to as a mutant.

organism     Any living thing, from elephants and plants to bacteria and other types of single-celled life.

programmable     A device or system that contains a computer, which allows the functions to change in a prescribed way, usually as determined by the user or manufacturer.

protein     A compound made from one or more long chains of amino acids. Proteins are an essential part of all living organisms. They form the basis of living cells, muscle and tissues; they also do the work inside of cells. Among the better-known, stand-alone proteins are the hemoglobin (in blood) and the antibodies (also in blood) that attempt to fight infections. Medicines frequently work by latching onto proteins.

RNA     A molecule that helps “read” the genetic information contained in DNA. A cell’s molecular machinery reads DNA to create RNA, and then reads RNA to create proteins.

sickle cell anemia     A disease where someone inherits an abnormal red-blood-cell-making gene from each parent. The red blood cells tend to be more rigid than normal and crescent — or “sickle” — shaped. As they flow through blood vessels, these cells don’t bend to slide through traffic jams, as normal blood cells do. Instead, they can get sticky and clog vessels, limiting blood flow and oxygen availability to tissues. In bad cases, sickled cells can cause painful “crises” requiring hospitalization for treatment.

technology     The application of scientific knowledge for practical purposes, especially in industry — or the devices, processes and systems that result from those efforts.

Citation

Journal: N.M. Gaudelli et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature. Published online October 25, 2017. doi: 10.1038/nature24644.

Journal: D.B.T. Cox et al. RNA editing with CRISPR-Cas13. Science. Published online October 25, 2017. doi: 10.1126/science.aaq0180.

Journal: G. Li et al. Highly efficient and precise base editing in discarded human tripronuclear embryos. Protein & Cell. Vol. 8, October 2017, p. 776. doi:10.1007/s13238-017-0458-7.

Journal: P. Liang et al. Correction of β-thalassemia mutant by base editor in human embryos. Protein & Cell. Published online September 23, 2017. doi: 10.1007/s13238-017-0475-6.