Genome editing, also called genome engineering or gene editing, is a type of genetic engineering that changes DNA in the genome of a living organism. Unlike older methods that randomly add genetic material to a genome, genome editing places changes in specific locations. The process uses tools that can recognize certain parts of DNA, cut the DNA at those spots, and then repair the cuts using one of two methods: homology-directed recombination or non-homologous end joining.

In 2015, the development of CRISPR gene editing made it easier, more accurate, and more practical to edit large numbers of genes.

Since 2015, scientists have tested genome editing on non-viable human embryos in experiments. In 2019, the first humans were born from genome-edited embryos using CRISPR, which happened during the controversial He Jiankui affair.

History

Genome editing began in the 1990s, before the development of modern gene-editing tools. At that time, editing was not very effective. In 2011, scientists used three types of engineered nucleases—zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and engineered meganucleases. These tools were named the 2011 Method of the Year by Nature Methods. The CRISPR-Cas system was named the 2015 Breakthrough of the Year by Science.

By 2015, four types of engineered nucleases were used: meganucleases, ZFNs, TALENs, and the CRISPR/Cas9 system. By 2017, nine different genome editors were available.

In 2018, scientists used engineered nucleases, sometimes called "molecular scissors," to make precise cuts in DNA. These cuts were repaired through two processes: nonhomologous end-joining (NHEJ) or homologous recombination (HR), which can create targeted changes in DNA.

In May 2019, lawyers in China reported that regulations were being drafted to hold people accountable for any harm caused by gene-editing techniques, such as CRISPR, after a scientist claimed to have created the first gene-edited humans (see Lulu and Nana controversy). Some experts have warned about possible risks of CRISPR and related technologies due to the unpredictable nature of cellular processes.

The University of Edinburgh Roslin Institute created pigs resistant to a virus that causes porcine reproductive and respiratory syndrome, a disease costing $2.6 billion annually in the US and Europe.

In February 2020, a US trial safely tested CRISPR gene editing on three cancer patients. In 2020, a tomato called Sicilian Rouge High GABA, which produces more of an amino acid linked to relaxation, was approved for sale in Japan.

In 2021, England (but not the rest of the UK) planned to remove restrictions on gene-edited plants and animals, shifting from European Union rules to regulations similar to those in the US and other countries. A 2021 report by the European Commission suggested that current rules for gene editing might not be suitable. Later in 2021, scientists introduced a CRISPR alternative called obligate mobile element–guided activity (OMEGA), which uses proteins like IscB, IsrB, and TnpB found in transposons and guided by small ωRNAs.

Background

Genetic engineering, a method used to add new genetic material to organisms, has been around since the 1970s. One problem with this technology is that DNA is often inserted randomly into the host’s genome, which can harm or change other genes in the organism. However, scientists have developed methods to insert genes into specific locations within the genome. These methods also allow for editing specific parts of the genome and reducing unintended effects. This can help in research by targeting specific genes for study and in gene therapy. By inserting a working gene into an organism and placing it where a faulty gene is, it may be possible to treat certain genetic diseases.

Early techniques to insert genes into specific locations in an organism’s genome (called gene targeting) used a process called homologous recombination (HR). Scientists created DNA pieces that matched the target area of the genome. The HR process in cells then inserted the DNA at the desired spot. Using this method on embryonic stem cells led to the creation of transgenic mice with specific genes turned off. Scientists could also add genes or change how genes are expressed. Mario Capecchi, Martin Evans, and Oliver Smithies were awarded the 2007 Nobel Prize for Physiology or Medicine for discovering how homologous recombination can be used to modify genes in mice through embryonic stem cells.

If a critical gene is turned off, it can be deadly to the organism. To study the function of such genes, scientists use site-specific recombinases (SSR). The most common types are the Cre-LoxP and Flp-FRT systems. Cre recombinase is an enzyme that removes DNA by recombining it between special sequences called Lox-P sites. The Flp-FRT system works similarly, with Flp recombinase recognizing FRT sequences. By breeding an organism with recombinase sites around the gene of interest with another organism that produces SSR only in certain cells, scientists can turn off or activate genes in specific cells. These techniques were also used to remove marker genes from transgenic animals. Later improvements allowed scientists to control when genes are turned off or activated, depending on the stage of development.

Process

A common way to edit DNA involves creating a double-stranded DNA break (DSB) and using the cell's natural repair processes. Two main repair methods exist: non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ connects DNA ends directly using enzymes, while HDR uses a similar DNA sequence as a template to fix the break. Scientists can use this process by designing a tool with the desired DNA changes inside a sequence that matches the DNA around the break. This allows the desired change to be added at the break site. HDR-based editing works similarly to homologous recombination, but it happens much faster.

Creating a precise DSB in the genome is essential for genome editing. Restriction enzymes can cut DNA but often target many sites. To make cuts at specific locations, scientists have developed three types of nucleases: Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALEN), meganucleases, and the CRISPR/Cas9 system.

Meganucleases, discovered in the 1980s, are enzymes that cut long DNA sequences (14 to 40 base pairs). The most common type is the LAGLIDADG family, named after a shared amino acid sequence. Meganucleases are naturally very specific because they recognize long DNA sequences. However, finding a meganuclease that matches a specific DNA sequence is difficult. Scientists have used methods like mutagenesis and high-throughput screening to create custom meganucleases. Others have combined different meganucleases to make hybrid enzymes that recognize new sequences. Some have altered the DNA-interacting parts of meganucleases to design tools that target specific sequences. Computer models are also used to predict how these modified meganucleases will work.

A large collection of protein units has been created to build chimeric meganucleases that recognize specific DNA sequences. These tools are used in research, health, agriculture, and industry. For example, two meganucleases have been developed to cut the human XPC gene, which is linked to Xeroderma pigmentosum, a rare condition that increases skin cancer risk from UV exposure.

Meganucleases cause less cell damage than methods like ZFNs, likely because they recognize DNA sequences more precisely. However, creating meganucleases for every possible DNA sequence is expensive and time-consuming, unlike methods such as ZFNs and TALENs, which use combinations of building blocks.

ZFNs and TALEN technology use a non-specific DNA-cutting part linked to proteins that recognize specific DNA sequences. Scientists first found an enzyme that cuts DNA separately from its recognition site. This enzyme’s cutting part was made non-specific and linked to proteins that can identify unique DNA sequences, increasing precision.

Zinc finger motifs are found in transcription factors, proteins that control gene activity. Zinc ions help shape these proteins’ structures, especially at sites where they interact with DNA. Each zinc finger recognizes a short DNA sequence (about 3 base pairs). Combining 6 to 8 zinc fingers allows recognition of longer sequences (around 20 base pairs). This enables scientists to control gene expression. For example, this method has been used to promote blood vessel growth in animals. By fusing zinc finger proteins with an enzyme’s cutting part, scientists can create tools to make precise DNA breaks.

The most common method uses two zinc finger proteins, each attached to a specific DNA sequence, linked to the FokI enzyme’s cutting part. The FokI enzyme needs to pair with another FokI unit to cut DNA. When the zinc finger proteins bind to their target sequences, the FokI units come together, allowing the enzyme to cut DNA. This increases precision because each zinc finger protein recognizes a unique sequence. Scientists have modified FokI to work only when paired with another FokI unit, further improving accuracy.

To design ZFNs for specific DNA sequences, scientists often combine known zinc finger units. Techniques using bacteria, yeast, or mammal cells help identify the best combinations. Studies show that ZFNs with 24 base pair recognition sites and modified FokI units create only one to two DNA breaks in cells, minimizing unintended effects.

Using FokI units that require pairing avoids unwanted activity from identical units, increasing precision. While ZFNs and TALENs both use similar cutting parts, they differ in how they recognize DNA. ZFNs use zinc fingers, while TALENs use TALE proteins. Zinc fingers repeat every three base pairs and are found in many proteins. Each zinc finger’s ability to bind DNA can be influenced by neighboring fingers. TALEs, however, match each amino acid to a specific DNA base pair. Both systems use repeated patterns, allowing scientists to create tools for many DNA sequences.

Precision and efficiency of engineered nucleases

The Meganucleases method of gene editing is the least effective compared to other methods. Because of how it binds to DNA and cuts it, it can only find one target in every 1,000 DNA building blocks. ZFN was created to fix this problem. It can find a target in every 140 DNA building blocks. However, both methods are hard to predict because their DNA-binding parts can interfere with each other. This means experts need a lot of time and money to test and confirm their results.

TALE nucleases are the most precise and specific method. They work better than Meganucleases and ZFN because their DNA-binding part is made of many TALE subunits. Each subunit can recognize a specific DNA piece on its own, allowing them to find more target sites with high accuracy. New TALE nucleases take about one week and a few hundred dollars to make, and they require skills in molecular biology and protein engineering.

CRISPR nucleases are slightly less precise than TALE nucleases. This is because CRISPR needs a specific DNA piece at one end to create the guide RNA it uses to repair DNA breaks. It is the fastest and cheapest method, costing less than $200 and taking only a few days. CRISPR also needs less expertise in molecular biology because the guide RNA, not the proteins, determines how it works. A major advantage of CRISPR is that it can target different DNA sequences using its short guide RNA, while ZFN and TALEN require designing and testing new proteins for each DNA sequence.

Because cutting DNA incorrectly could cause harm at the genetic or organism level, improving the accuracy of Meganucleases, ZFNs, CRISPR, and TALEN-based tools has been a major research focus. Studies show ZFNs often cause more cell damage than TALEN or CRISPR methods. TALEN and CRISPR methods usually work more efficiently and have fewer mistakes in targeting the wrong DNA spots. Based on how far apart the DNA-binding and cutting parts are, TALEN methods achieve the highest precision.

Multiplex Automated Genomic Engineering (MAGE)

Before the development of new technology, scientists had a very slow, costly, and inefficient way to study how genes affect traits. Researchers used to change one gene at a time, observe the result, and repeat the process with a different gene. To solve this problem, scientists at the Wyss Institute at Harvard University created MAGE, a tool that makes it faster and easier to edit genomes inside living cells. MAGE works in a small machine that fits on a kitchen table. It combines changes made by scientists with natural changes that happen when cells divide, creating many different genetic variations.

Scientists use synthetic single-stranded DNA (ssDNA) and a group of short DNA pieces called oligonucleotides to make changes in specific parts of a cell’s genome. The process starts by using a method called electroporation to introduce ssDNA into cells. Then, the cells grow, and proteins from bacteriophages help the ssDNA attach to the cell’s DNA. Scientists test for changes by growing cells on special media that show specific traits. Each step takes about 2.5 hours, and more time is needed to grow and analyze the cells. By repeatedly adding many different ssDNAs that target multiple areas of the genome, MAGE can create many genetic changes in a group of cells. This process can make up to 50 changes at once, including small single-letter changes or large changes across entire genes or networks, all within a few days.

MAGE experiments are grouped into three types based on how many genes or changes are involved: (i) many target sites with one change each, (ii) one target site with many changes, and (iii) many target sites with many changes. An example of type (iii) was in 2009, when scientists used MAGE to program Escherichia coli bacteria to produce five times more lycopene, a substance found in tomato seeds that helps fight cancer. They used MAGE to improve a chemical pathway in the bacteria to make more lycopene. This experiment took about 3 days and cost just over $1,000 in materials. The speed, simplicity, and low cost of MAGE can change how industries make important products in fields like medicine, energy, agriculture, and chemical manufacturing.

Applications

As of 2012, effective genome editing had been developed for many experimental systems, including plants and animals, often beyond what is needed for medical purposes. It was becoming a common method used in research labs. Recent examples include creating mutant rats, zebrafish, maize, and tobacco using ZFN technology. Improvements in TALEN-based methods also show how important these techniques are, and the list of uses is growing quickly. Genome editing with engineered nucleases will likely help many areas of life sciences, such as studying how genes work in plants and animals and developing gene therapies for humans. For example, synthetic biology, which aims to design cells and organisms to perform new tasks, can benefit from the ability to add or remove parts of the genome using engineered nucleases. Scientists can also study gene functions using stem cells with these tools.

This method can perform the following tasks:

• Changing specific genes
• Treating genetic diseases
• Creating changes in chromosomes
• Studying gene functions using stem cells
• Producing transgenic animals
• Labeling genes within an organism
• Adding new genes to specific locations

Recent advances in genetic engineering, especially gene editing and improvements in bovine reproduction methods like in vitro embryo culture, allow scientists to edit genomes directly in fertilized eggs using synthetic endonucleases. A new tool called CRISPR/Cas9, which uses RNA to guide endonucleases, has expanded the range of available methods. CRISPR/Cas9 allows scientists to use multiple guide RNAs to make several changes at once in mammalian zygotes through a process called cytoplasmic direct injection.

Gene editing can also be used in certain fish species in aquaculture, such as Atlantic salmon. While this is still experimental, it could lead to fish with traits like faster growth, disease resistance, sterility, controlled reproduction, and specific colors. Selecting these traits may help create a more sustainable environment and improve fish welfare.

AquAdvantage salmon is a genetically modified Atlantic salmon developed by AquaBounty Technologies. In this salmon, the gene that controls growth hormone activity has been replaced with a similar gene from the Pacific Chinook salmon and a promoter sequence from the ocean pout.

Thanks to progress in single-cell transcriptomics, genome editing, and new stem cell models, scientists can now study gene functions directly in human samples, not just in animal models. Single-cell gene expression analysis has identified key genes involved in human development, which are being tested for their roles. Using data from global transcriptomics, CRISPR-based tools can now be used to remove or disable important genes in human cells to study their functions.

Genome editing with Meganuclease, ZFNs, and TALEN provides new ways to change genes in plants. These methods may help create plants with desired traits by modifying their own genes. For example, adding specific genes to major crops can link multiple traits together to ensure they are passed on during breeding. Recent studies in Arabidopsis thaliana and Zea mays (corn) show that ZFNs can introduce herbicide-resistant genes into plants. In Arabidopsis, two herbicide-resistant genes were added to specific locations with up to 2% of cells showing successful changes. In corn, ZFNs caused targeted gene changes, and these changes were passed on to future generations. A successful example of genome editing in crops is the use of CRISPR/Cas9 to disable a virus in bananas, helping solve a major challenge in banana farming.

TALEN-based methods have been tested and improved for use in plants. A U.S. food company, Calyxt, used TALEN to improve soybean oil quality and increase potato storage potential.

To improve genome editing in plants using ZFNs, several challenges need to be addressed. These include designing reliable nucleases, ensuring they are not harmful, choosing the right plant tissues for editing, controlling how enzymes work, avoiding unintended changes in other genes, and accurately detecting successful edits.

A common way to deliver CRISPR/Cas9 in plants is through Agrobacterium-based transformation. This method uses a system called T4SS to insert genetic material directly into plant cells. Scientists create plasmids containing Cas9 and guide RNA, which are then transferred into Agrobacterium for use in plants. To improve delivery in living plants, scientists are exploring viruses as more effective tools for transferring genetic material.

Research

The best way to use gene therapy is to replace a faulty gene with a healthy version in its usual place. This method is better than using viruses to deliver genes because it does not require the full instructions or control sections of the gene when only a small part needs to be changed, which is common in many cases. The altered genes also work more naturally within cells compared to full genes carried by viruses.

In 2015, scientists first used a technique called TALEN-based genome editing to treat CD19+ acute lymphoblastic leukemia in an 11-month-old child. They modified donor T cells to attack the cancer cells, resist a drug called Alemtuzumab, and avoid being detected by the patient’s immune system after being introduced into the body.

Scientists have tested CRISPR-Cas9 in cells and animals to fix genetic mistakes that cause diseases such as Down syndrome, spina bifida, anencephaly, and Turner and Klinefelter syndromes.

In February 2019, researchers working with Sangamo Therapeutics, a company in Richmond, California, announced the first human treatment that permanently changes DNA inside the body. This therapy was used on a patient with Hunter syndrome. Sangamo is currently testing gene-editing treatments using a method called Zinc Finger Nuclease (ZFN).

Scientists have used CRISPR-Cas9 to change genes linked to sterility in A. gambiae, a type of mosquito that spreads malaria. This method could also help reduce other diseases carried by insects, such as yellow fever, dengue, and Zika.

The CRISPR-Cas9 system can be programmed to control the number of bacteria by targeting specific types. It can help good bacteria grow while removing harmful ones, which is more effective than using broad-spectrum antibiotics.

Research is ongoing to use CRISPR for treatments against human viruses like HIV, herpes, and hepatitis B. Scientists are studying ways to target the virus or the human cells it infects to stop the virus from entering cells. In November 2018, a scientist named He Jiankui claimed to have edited the genes of two human embryos to disable the CCR5 gene, which helps HIV enter cells. He said twin girls, Lulu and Nana, were born a few weeks later. However, the girls still had working copies of the CCR5 gene, meaning they were not fully protected from HIV. This work was criticized as unethical, dangerous, and too early.

In January 2019, scientists in China reported creating five genetically modified monkeys that are exact copies of each other. They used the same cloning method that produced the first cloned monkeys, Zhong Zhong and Hua Hua, and the first cloned sheep, Dolly. They also used the same CRISPR-Cas9 gene-editing technique that He Jiankui reportedly used to modify human embryos. These monkeys were created to study medical conditions.

Prospects and limitations

In the future, an important goal of research into genome editing with special tools must be to improve the safety and accuracy of these tools. For example, better ways to find unintended changes can help scientists learn how to prevent them. ZFNs sometimes cause unintended effects, but changes to their structure can reduce these issues. Research by Dana Carroll shows that scientists need to understand more about how DNA repairs itself. In the future, one way to find other DNA changes caused by ZFNs might be to collect broken DNA pieces from cells and use high-speed sequencing to study them.

CRISPR is popular because it is easier to use and less expensive than other methods. There are more scientific papers about CRISPR than ZFNs or TALENs, even though CRISPR was discovered more recently. Both CRISPR and TALENs are preferred for large projects because they work well and are precise.

Genome editing also happens naturally in living things. Viruses and small RNA particles can change genetic material without human help.

Although DNA-based methods are better than some other techniques for studying genetics, they are still not very efficient. Often, less than half of treated cells get the desired changes. For example, when scientists use a cell’s repair system to create a mutation, another repair system might fix the DNA instead, making the mutation less likely.

Mice have been the most common animals for studying diseases. CRISPR can help connect mouse models to human medicine by creating disease models in larger animals like pigs, dogs, and monkeys. Using CRISPR-Cas9, scientists can directly introduce tools into early-stage animal embryos to make genetic changes. This skips a complicated step in creating new animal models, reducing the time needed by about 90%.

CRISPR’s power makes it useful for xenotransplantation, which is using animal organs in humans. Studies have shown CRISPR can remove harmful viruses from animal cells, lowering the risk of disease and immune rejection. This makes using animal organs in humans more likely.

In plants, genome editing can help protect biodiversity. Gene drives might be used to control invasive species, but there are risks involved.

Some people believe genome editing could improve human traits. Australian scientist David Andrew Sinclair says new technologies could allow parents to have healthier children. A 2016 report suggested it might be possible to use genes from other organisms or synthetic genes to improve traits like night vision or smell. Scientist George Church has listed possible genetic changes, such as reducing the need for sleep or improving memory, along with possible risks.

In 2017, the American National Academy of Sciences and Medicine supported human genome editing under strict rules. They said clinical trials might be allowed in the future only for serious conditions after safety and efficiency issues are solved.

Risks

In 2016, the United States Director of National Intelligence, James R. Clapper, stated in the Worldwide Threat Assessment that genome editing could be used as a weapon of mass destruction. He noted that countries with rules or ethical standards different from Western countries might increase the risk of creating harmful biological agents or products. The report explained that the widespread availability, low cost, and fast development of this technology could lead to misuse, either intentionally or by accident, which might cause major economic and national security problems. For example, tools like CRISPR could be used to create "killer mosquitoes" that spread diseases and destroy crops.

A 2016 report by the Nuffield Council on Bioethics said that the ease and low cost of genetic editing tools could allow people without formal training—called "biohackers"—to conduct experiments, which might lead to the release of genetically modified organisms. The report also highlighted that changing a person’s genome in a way that affects future generations is very complex and needs careful ethical review. These changes could cause unexpected harm to the person and their future children, as the altered genes could be passed through sperm or eggs. In 2001, Australian researchers Ronald Jackson and Ian Ramshaw faced criticism for publishing a study in the Journal of Virology that explored using an altered mousepox virus to control mice, a major pest in Australia. The study was criticized because it might help bioterrorists create biological weapons, such as vaccine-resistant strains of viruses like smallpox, which could harm humans. Additionally, there are concerns about the risks to ecosystems from releasing gene drives into wild populations.

Nobel prize

In 2007, the Nobel Prize for Physiology or Medicine was awarded to Mario Capecchi, Martin Evans, and Oliver Smithies for developing methods to change specific genes in mice using embryonic stem cells.

In 2020, the Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer Doudna for creating a technique to make precise changes to DNA.