Genome editing

Genome editing, or genome engineering, 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 inserts genetic material into a host genome, genome editing targets the insertions to site specific locations. Genome editing, or genome engineering, 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 inserts genetic material into a host genome, genome editing targets the insertions to site specific locations. In 2018, the common methods for such editing use engineered nucleases, or 'molecular scissors'. These nucleases create site-specific 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'). As of 2015 four families of engineered nucleases were used: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system. Nine genome editors were available as of 2017. Genome editing with engineered nucleases, i.e. all three major classes of these enzymes—zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and engineered meganucleases—were selected by Nature Methods as the 2011 Method of the Year. The CRISPR-Cas system was selected by Science as 2015 Breakthrough of the Year. 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. Genetic engineering as a 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 hosts genome. This can impair or alter other genes within the organism. Methods were sought which targeted the inserted genes to specific sites within an organism genome. As well as reducing off-target effects it also enabled the editing of specific sequences within a genome. 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. Early methods to target genes to certain sites within a genome (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. 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. 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.