Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly insert genetic material into a host genome, genome editing targets the insertions to site-specific locations.
Programmable molecules called restriction endonucleases, also known as gene editors or molecular scissors, are used to create double-strand breaks (DSBs) at desired locations in the genome. The induced breaks are repaired through homology-directed recombination (HDR) or non-homologous end joining (NHEJ), resulting in targeted mutations.
The development of CRISPR gene editing in 2015 improved the efficiency, specificity, and practicality of large-scale genome editing.
One of the earliest and most efficient methods of geonome editing is base editing. It does not require breaking and recombining the genomic DNA strands, and thus avoids the random insertions and deletions associated with these procedures. However, it is only appropriate for precise editing of single nucleotides. It employs nucleobase modifying enzymes directed by nucleic acid guide sequences. It was first described in the 1990s and has seen a resurgence more recently.
Since 2015, genome editing has been experimentally investigated on non-viable human embryos. In 2019, the first humans were born from genome-edited embryos using the CRISPR technique, as a result of the controversial He Jiankui affair.
History Genome editing was pioneered in the 1990s, before the advent of the common current nuclease-based gene-editing platforms, but its use was limited by low efficiencies of editing.
In the 2000s, genome editing with engineered nucleases (also known as "molecular scissors") was introduced. , three families of engineered nucleases were used: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN). All of them were selected by Nature Methods as the 2011 Method of the Year. In 2015 the CRISPR-Cas system was introduced, and it was selected by Science as 2015 Breakthrough of the Year. Nine genome editors were available .
In 2018, the common methods for such editing used engineered nucleases to create double-strand breaks (DSBs) at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous end-joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations ('edits').
In May 2019, lawyers in China reported, in light of the purported creation by Chinese scientist He Jiankui of the first gene-edited humans (see Lulu and Nana controversy), the drafting of regulations that anyone manipulating the human genome by gene-editing techniques, like CRISPR, would be held responsible for any related adverse consequences. A cautionary perspective on the possible blind spots and risks of CRISPR and related biotechnologies has been recently discussed, focusing on the stochastic nature of cellular control processes.
The University of Edinburgh Roslin Institute engineered pigs resistant to a virus that causes porcine reproductive and respiratory syndrome, which costs US and European pig farmers $2.6 billion annually.
In February 2020, a US trial safely showed CRISPR gene editing on 3 cancer patients. In 2020 Sicilian Rouge High GABA, a tomato that makes more of an amino acid said to promote relaxation, was approved for sale in Japan.
In 2021, England (not the rest of the UK) planned to remove restrictions on gene-edited plants and animals, moving from European Union-compliant regulation to rules closer to those of the US and some other countries. An April 2021 European Commission report found "strong indications" that the current regulatory regime was not appropriate for gene editing. Later in 2021, researchers announced a CRISPR alternative, labelled obligate mobile element–guided activity (OMEGA) proteins including IscB, IsrB and TnpB as endonucleases found in transposons, and guided by small ωRNAs.
In 2026, Zilong Qiu and his team at the Shanghai Jiao Tong University School of Medicine, published successful use of base editing in mice and monkeys to induce corrected CHD3 gene that causes Snijders Blok–Campeau syndrome. It was subsequently discovered that a year before, Qiu had treated a six-year-old girl at Xinhua Hospital in Shanghai using engineered viruses to carry the corrected gene into the spinal fluid. The girl died of thrombotic microangiopathy several days later.
Background Genetic engineering, as method of introducing new genetic elements into organisms, has been around since the 1970s. One drawback of this technology has been the random nature with which the DNA is inserted into the host's genome, which can impair or alter other genes within the organism. However, several methods have been discovered that target the inserted genes to specific sites within an organism's genome. It has also enabled the editing of specific sequences within a genome, as well as reduced off-target effects. This could be used for research purposes, by targeting mutations to specific genes, and in gene therapy. By inserting a functional gene into an organism, and targeting it to replace the defective one, it could be possible to cure certain genetic diseases.
Gene targeting
Homologous recombination Early methods to target genes to certain sites within a genome of an organism (called gene targeting) relied on homologous recombination (HR). By creating DNA constructs that contain a template that matches the targeted genome sequence, it is possible that the HR processes within the cell will insert the construct at the desired location. Using this method on embryonic stem cells led to the development of transgenic mice with targeted genes knocked out. It has also been possible to knock in genes or alter gene expression patterns. In recognition of their discovery of how homologous recombination can be used to introduce genetic modifications in mice through embryonic stem cells, Mario Capecchi, Martin Evans and Oliver Smithies were awarded the 2007 Nobel Prize for Physiology or Medicine.
Conditional targeting If a vital gene is knocked out, it can prove lethal to the organism. In order to study the function of these genes, site specific recombinases (SSR) were used. The two most common types are the Cre-LoxP and Flp-FRT systems. Cre recombinase is an enzyme that removes DNA by homologous recombination between binding sequences known as Lox-P sites. The Flip-FRT system operates in a similar way, with the Flip recombinase recognising FRT sequences. By crossing an organism containing the recombinase sites flanking the gene of interest with an organism that express the SSR under control of tissue specific promoters, it is possible to knock out or switch on genes only in certain cells. These techniques were also used to remove marker genes from transgenic animals. Further modifications of these systems allowed researchers to induce recombination only under certain conditions, allowing genes to be knocked out or expressed at desired times or stages of development.
Process
Double strand break repair
A common form of genome editing relies on the concept of DNA double stranded break (DSB) repair mechanics. There are two major pathways that repair DSB; non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ uses a variety of enzymes to directly join the DNA ends, while the more accurate HDR uses a homologous sequence as a template for regeneration of missing DNA sequences at the break point. This can be exploited by creating a vector with the desired genetic elements within a sequence that is homologous to the flanking sequences of a DSB. This will result in the desired change being inserted at the site of the DSB. While HDR based gene editing is similar to the homologous recombination based gene targeting, the rate of recombination is increased by at least three orders of magnitude.
Engineered nucleases
The key to genome editing is creating a DSB at a specific point within the genome. Commonly used restriction enzymes are effective at cutting DNA, but generally recognize and cut at multiple sites. To overcome this challenge and create site-specific DSB, four distinct classes of nucleases have been discovered and bioengineered to date: Meganucleases Zinc finger nucleases (ZFNs) Transcription-activator like effector nucleases (TALEN) Clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system.
Meganucleases
Meganucleases, discovered in the late 1980s, are enzymes in the endonuclease family which are characterized by their capacity to recognize and cut large DNA sequences (from 14 to 40 base pairs). The most widespread and best known meganucleases are the proteins in the LAGLIDADG family, which owe their name to a conserved amino acid sequence.
Meganucleases, found commonly in microbial species, have the unique property of having very long recognition sequences (>14bp) thus making them naturally very specific. However, there is virtually no chance of finding the exact meganuclease required to act on a chosen specific DNA sequence. To overcome this challenge, mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences. Others have been able to fuse various meganucleases and create hybrid enzymes that recognize a new sequence. Yet others have attempted to alter the DNA interacting aminoacids of the meganuclease to design sequence specific meganucleases in a method named rationally designed meganuclease. Another approach involves using computer models to try to predict as accurately as possible the activity of the modified meganucleases and the specificity of the recognized nucleic sequence.
A large bank containing several tens of thousands of protein units has been created. These units can be combined to obtain chimeric meganucleases that recognize the target site, thereby providing research and development tools that meet a wide range of needs (fundamental research, health, agriculture, industry, energy, etc.) These include the industrial-scale production of two meganucleases able to cleave the human XPC gene; mutations in this gene result in Xeroderma pigmentosum, a severe monogenic disorder that predisposes the patients to skin cancer and burns whenever their skin is exposed to UV rays.
Meganucleases have the benefit of causing less toxicity in cells than methods such as Zinc finger nuclease (ZFN), likely because of more stringent DNA sequence recognition; however, the construction of sequence-specific enzymes for all possible sequences is costly and time-consuming, as one is not benefiting from combinatorial possibilities that methods such as ZFNs and TALEN-based fusions utilize.
Zinc finger nucleases
As opposed to meganucleases, the concept behind ZFNs and TALEN technology is based on a non-specific DNA cutting catalytic domain, which can then be linked to specific DNA sequence recognizing peptides such as zinc fingers and transcription activator-like effectors (TALEs). The first step to this was to find an endonuclease whose DNA recognition site and cleaving site were separate from each other, a situation that is not the most common among restriction enzymes. Once this enzyme was found, its cleaving portion could be separated which would be very non-specific as it would have no recognition ability. This portion could then be linked to sequence recognizing peptides that could lead to very high specificity.