Gene Editing - Genome Editing 1-2


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    Gene Editing

    Gene Editing

    What is Gene Editing?

    Definition: Gene editing (or genome editing) is the insertion, deletion or replacement of DNA at a specific site in the genome of an organism or cell. It is usually achieved in the lab using engineered nucleases also known as molecular scissors

  • Guide to Gene Editing
  • How Gene Editing Works
  • Applications of Gene Editing
  • Gene Editing Resources
    • Guide to Gene Editing

    Gene Editing Techniques

    There are a number of recognised gene editing methods. Editing the genome can be achieved using engineered nucleases such as
    For the most part, gene editing companies can separate genome modifications into one of two experimental categories:
    ■Loss of function - functional forms of the genome are removed from the system and the effect studied
    ■Gain of function - active (often mutant) forms of the genome are introduced into the system and the effect studied
    Up until recently most loss and gain of function analyses was performed using RNAi and transgenesis respectively - both enormously powerful techniques, but they do have limitations.

    Transgene Overexpression

    Transgene overexpression can lead to artefacts that are a consequence of that overexpression - and this is especially true in the case of oncogenes, where their over-abundance can lead to transformative effects that would not normally be seen in a physiological system.
    Retroviral Overexpression
    ■Large growth induction phenotype
    ■Transforming alone
    Gene editing: How single gene knock-in removes false positives with MCF10As
    ■Milder growth induction phenotype
    ■Non-transforming alone
    Overexpression of oncogenes can over represent their role in disease biology.
    Genome editing allows scientists to perform the same types of loss and gain of function experiments, but manipulate genes of interest at the endogenous level. So for loss of function, the gene can be rendered non-functional or completely removed from the system. For gain of function, mutations or reporter tags can be expressed from the promoter of the gene itself.
    How Gene Editing Works

    How Gene Editing Works

    There are now a number of tools available to scientists interested in performing genome editing experiments, which can be split into two categories: engineered nucleases and recombinant adeno-associated virus (rAAV).
    Engineered nucleases generate gene edits by introducing double strand breaks (DSBs) at the target site, and stimulating the cells own DNA repair pathways to make modifications.
    DSBs will most frequently be repaired by non-homologous end joining (NHEJ), which is error prone and can result in the introduction of insertions or deletions. In this manner frameshift mutations can be introduced into the coding sequence of genes.
    If the DSB is introduced in the presence of a homologous donor sequence then in some cases repair will occur via the homology-directed repair (HR) pathway. In this manner modifications and exogenous sequence can be introduced at the target site. Altering genomic sites using HR is often referred to as gene targeting (although engineered nucleases are not always used).
    There are three commonly used nucleases available to gene editors:
    rAAV on the other hand relies solely on the HR pathway for making modifications. It essentially functions as a highly efficient means of delivering donor DNA into the nuclei of cells, where in some cases it will be integrated into the genome by HR.
    The advantage of rAAV versus nuclease based technologies that no cleavage event occurs, which means that there is a lower risk of uncontrolled off-target modifications. The disadvantage of rAAV versus nucleases is that it is lower efficiency (often <1% efficiency), meaning that much more clones have to be screened to find one that is correctly targeted.
    Applications of Gene Editing

    Applications of Gene Editing

    As discussed above, many scientists use gene editing technology for loss or gain of function analyses. These approaches can be used to address a huge variety of questions - not least the role that a specific gene or mutation plays in a biological pathway or the pathogenesis of a disease. And in much the same way that scientists have done with preceeding technologies such as RNAi and transgenesis, a wide variety of read outs, such as expression, localisation, interaction and pathway activation, can be used to study the effect of the gene edit.

    Examples of Gene Editing Uses