By Andrew Kueh, Head, Melbourne Advanced Gene Editing Centre (MAGEC) Platform Lead; Maggie Potts, Postdoctoral Research Fellow, Olivia Newton-John Cancer Research Institute; Emily Lelliott, Postdoctoral Research Fellow, Olivia Newton-John Cancer Research Institute
The ability to modify and edit genomic material has been crucial to expanding our understanding of disease progression. It is used in developing novel applications within industry, such as gene/immunotherapy interventions and identifying cancer vulnerabilities through large-scale whole genome screens.
The gene editing process is highly adaptable and can be used to generate several precise outcomes depending on the intended application. For instance, this can be executed in the following ways:
- The deletion and removal of a specific stretch of genomic material
- The insertion of exogenous genomic sequences into a particular locus, and
- The complete replacement of a length of genomic material with a new sequence.
In the recent years, Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) has revolutionised the efficiency, speed and simplicity of gene editing.
Originally discovered in the bacteria Streptococcus pyogenes, as part of an elegant defence system against invading viruses, scientists unravelled the mechanism of the CRISPR/Cas9 protein and its ability to act as “molecular scissors” cutting through double stranded DNA at a defined genomic location pre-determined by a guide RNA. These double-stranded breaks can result in the removal of gene sequences, or the insertion or replacement of gene sequences if a DNA repair template is also introduced.
Using genome editing to find disease-causing mutations
Our work at the Melbourne Advanced Genome Editing Centre (MAGEC) has led to the development of >450 gene modified mouse models, of which several have been used in industry-linked projects.
An example of our work, previously published in Nature1, saw a group of patients from three families first present with symptoms of episodic high fevers and swollen lymph nodes to be found related to a new autoinflammatory disease known as CRIA syndrome.
Deep sequencing these patients’ cells revealed mutations all aligning within the gene RIPK1 at the same genomic location. These patient mutations all happen to block cleavage of RIPK1, which disrupts its essential function in regulating inflammation.
The MAGEC facility generated gene modified pre-clinical mouse models using CRISPR/Cas9 that replicate the exact patient mutations within RIPK1. These models subsequently developed the same autoinflammatory symptoms, confirming that these were indeed the disease-causing mutations.
These pre-clinical models also facilitated the development and optimisation of new CRIA patient specific therapies such as anti-inflammatory medications and RIPK1 inhibitors, prior to use in clinical trials.
This exemplifies the potential of this technology in developing personalised therapies and medicines.
As part of the Genome Engineering and Cancer Modelling Program at the Olivia Newton-John Cancer Research Institute (ONJCRI), we also conduct functional genomic screens in cancer cell lines, primary immune cells, and mouse models to study tumour biology and immunology.
Over the past decade, advancements in CRISRP/Cas9 technologies have enabled not only gene deletions and insertions, but also a range of modifications including modulation of gene expression ‘on’ or ‘off’, termed CRISPR activation or inhibition respectively, and precise single base modifications, called CRISPR base editing.
Our laboratory has established CRISPR-enabled mouse models that we use with specific cancer disease models to identify which genes promote tumour onset and which genes confer therapeutic resistance.
For example, using our novel CRISPR activation mouse enabled us to develop a new model of aggressive double hit lymphoma highly sensitive to the BH3 mimetic drug Venetoclax2.
By performing screens in vitro with our double hit lymphoma cell lines, we identified mechanisms of resistance to Venetoclax2. Furthermore, we are now using our CRISPR mouse models to identify genes that modulate immune cell behaviour and anti-cancer function.
Together, our CRISPR models and functional genomic screening approaches can be used to investigate various aspects of cancer and immune cell biology, ultimately informing therapeutic strategies that encompass both tumour-targeted therapies and cancer immunotherapies.
To learn more, visit MAGEC Platform.
