Our group has a long-term interest in exploring and exploiting the process of gene targeting in human cells. Gene targeting (also called genome editing) makes defined changes to the genome by harnessing homologous recombination (HR), one of the cellular pathways for repairing DNA double strand breaks (DSBs) and is greatly stimulated by customised nucleases (ZFNs, TALENs, CRISPRs) that specifically cleave the target locus.  Defined genomic changes are made when the resulting DSB is repaired by undergoing HR with a homologous DNA template co-delivered with the nuclease. Because it can be used to correct disease-causing mutations, gene targeting is a very attractive approach to gene therapy for inherited genetic disorders, including haematological diseases such as β-thalassemia major (βTM) and haemophilia. Along with the competing process of nonhomologous end-joining (NHEJ), gene targeting is also used to introduce mutations in order to study gene function, or potentially for therapeutic purposes. To promote the desired outcome in the appropriate cell type with sufficient specificity and efficiency, the key parameters in DSB repair must be identified and optimised. Such knowledge is also important for understanding factors that determine genome instability. Our projects fall into three main categories:

Investigating the underlying process of DNA double strand break repair

In previous work we have investigated the influence on gene targeting of factors such as chromosomal position, DNA delivery method and overexpressed recombination proteins such as Rad51 and Redβ. We have recently developed a natural reporter system for nuclease-induced DSB repair at the X-chromosomal HPRT locus by NHEJ and HR. We are using this system to further explore parameters affecting DSB repair and gene targeting, including the nature of the repair template and DSB.

Gene disruption for functional analyses and oncogene inactivation

After pioneering homozygous gene disruption in human cells (ISG12) we developed the first conditional gene knockouts in human cells (CDK1, TOPO II alpha), and studied their pleiotropic effects on cell cycle control, polyploidy, chromosome condensation/segregation and DSB repair. More recently we have investigated the role of the BCL6 oncogene in B cell growth and differentiation and have developed custom endonucleases for inactivating the BCR-ABL oncogene in Chronic Myeloid Leukemia cells.

Promoting gene correction at the β-globin locus (HBB)

With the long-term goal of gene correction therapy for β-haemoglobinopathies, such as βTM, we have used a customised endonuclease to promote efficient gene targeting at the HBB locus in cell lines. Our current goals are to investigate HBB gene correction in adult stem cells using HBB-specific CRISPR nucleases, and to explore alternative endonuclease and donor repair template designs.