Gene Editing

Gene Editing

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 achieved using engineered nucleases also known as molecular scissors

At Horizon, we've been working on genome editing technologies for over a decade and have licensed and developed a number of optimised solutions to enable research organisations and drug discovery companies using our innovative and high precision gene editing products and services to have confidence in their project outcomes.

Below are a selection of frequently asked questions and useful resources for those embarking on genome editing projects:

What is Gene Editing?

What is Gene Editing?

Editing the genome can be achieved with engineered nucleases such as CRISPR-Cas9, ZFNs or TALENs, or with viral systems such as rAAV. It has been used effectively in a wide variety of tissues and organisms – at Horizon, we perform genome editing through cell line engineering in human cancer cells and IPSCs, and also in mammalian model organisms such as knockout mice and rats.

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 both with limitations. The potential for off target effects with RNAi are well known, and challenges associated with incomplete knockdown and reproducibility add a further layer of complexity. 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.

Watch our introduction to gene editing

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 elicit 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.

Learn how we're combining rAAV, CRISPR and ZFNs for better gene editing

Gene Editing Uses

Gene Editing Uses

As discussed above, many scientists use genome editing technologies 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.

Here's a few interesting examples of how it has been used:

Learn more about isogenic cell line applications

Gene Editing Guide

A Guide to Gene Editing

At Horizon, we’d love to do everyone’s gene editing for them, and we have a range of products and services to suit different scientific needs, timelines and budgets.

But we also know how immensely powerful genome editing technologies can be, and how bringing the capability in house can be desirable. If you can't find what you need amongst our ready engineered cell lines and rat models, then we can supply cell line validated gRNAs and microinjection ready CRISPR reagents to help you hit the ground running, and below you'll find some resources to help get you started.

Genome Engineering Systems

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