Genome editing involves making desired changes to the sequence of a pre-determined DNA sequence of interest (the target site) at some specific location within the genome of an intact cell. (Because the target DNA sequence is usually a gene, genome editing has often been described as gene targeting.)
At its most fundamental level, genome editing relies on two components: (1) endo nucleases to cut both DNA strands at, or close to, a pre-determined target sequence of interest; and (2) protein complexes that rejoin the broken DNA strands. Following genome editing, screening must be carried out to identify those cells where a desired DNA change has been produced. There must, of course, be some way of ensuring a high degree of sequence specificity so that cleavage is directed to occur at a desired target sequence. And genome editing often (but not always) relies on artificial intervention to direct the desired sequence changes.
Two quite different genome editing approaches have been used (Figure 1). A traditional method, which has been used in mammalian cells for more than three decades, relies exclusively on homologous recombination using endogenous endonucleases that are part of the cell’s recombination machinery. The method relies on designing the transgene to have long stretches of DNA that show 100% sequence identity to the target site plus some altered sequence that can be introduced into the target site by recombination. The high degree of sequence homology between the transgene and the target site within the genome promotes recombination between them, in which case endogenous endonucleases cleave the DNA at, or close to, the target site (Figure 1A). We describe the principle of using standard homologous recombination in genome editing in Section 8.3. Homologous recombination can also be used to direct specific large-scale changes at pre-determined positions in the genome, including very large deletions, large-scale inversions, and translocations (chromosome engineering).
Fig1. Two very different approaches to genome editing. (A) Genome editing using homologous recombination (HR) alone. The transgene is designed to have some flanking sequences that are identical to those at a specific target site in the genome but altered sequence (yellow circles) in a central region. Cells are screened to identify rare recombination events between the transgene and target site, notably a double crossover that inserts the altered sequence into the target site (crossover points are indicated by a red X; yellow triangles signify cleavage points). Recombination is carried out by endogenous enzymes, including an endonuclease that cuts double-stranded DNA and a DNA ligase that rejoins the DNA fragments. (B) Genome editing using programmable exogenous nucleases. Introduced transgenes produce endonucleases plus protein or RNA guide sequences designed to bind to specific DNA sequences at a pre-determined target site in the genome. The exogenous endonuclease is covalently bound to a protein guide sequence or noncovalently to a hybrid RNA that contains a guide sequence plus binding sites for the endonuclease. A pair of sequence-specific guide sequences is used to target bound endonuclease, to cut both DNA strands at a desired unique site. The resulting double-strand break can be repaired rapidly, in which case errors are introduced that may introduce desired sequence changes (yellow circles), or by manipulating an homologous recombination pathway to introduce the desired changes. Different cells may have different altered sequences and are screened for the presence of a desired sequence alteration.
A second, quite recently developed method of genome editing uses programmable site-specific nucleases. Transfected transgenes are expressed to make exogenous endo nucleases plus protein or RNA guide sequences. The guide sequences are designed to bind to specific sequences at a pre-determined target site within the genome, and are physically bound in some way to the introduced endonuclease. With the exogenous endonucleases attached, the guide sequences bind to their target sequences whereupon the bound endonucleases cut both DNA strands to make a double break (Figure 1B).
The resulting double-strand DNA break activates an emergency DNA repair system (unnatural double-strand breaks in DNA can be fatal for a cell—the priority is to effect repairs rapidly). The method then relies on cellular DNA repair systems to repair the broken DNA. Often an error-prone DNA repair mechanism is used, which by itself introduces a desired change in the target DNA sequence. It is also possible to artificially intervene in the DNA repair to direct desired changes in the target DNA. In either case, treated cells are screened to identify those cells where the altered nucleotides are of the desired type.
Genome editing allows all manner of changes to be introduced into target sequences, but it can take some time. Rather than inactivate a gene, it is often more convenient to simply down-regulate the gene. Gene silencing is a fast and simple approach in which RNA transcripts of a pre-determined target gene are targeted to suppress gene expression.
Finally, Transgenes can be inserted into the germ line to make transgenic animals. That often involves first transfecting cultured pluripotent cells (embryonic stem cells or induced pluripotent stem cells) or germ-line precursor cells. From the perspective of human genetics, transgenic animals have three particularly valuable research applications, as listed below.
• Investigation of how mammalian (and animal) genes function (to allow understanding of different aspects of cell, tissue, and organ biology).
• Disease modeling. genetically modified animals are crucially important as models of human diseases, allowing deep insights into the molecular basis of disease.
• As test systems for proposed new disease treatments, especially where genetically modified animals have been shown to be good disease models.
