How Homing Endonucleases can be used to cut DNA

The Stoddard Lab at the Fred Hutchinson Cancer Research Center in Seattle, WA uses a combination of biochemistry, structural biology, and protein engineering to create custom-specificity enzymes which cleave genomic DNA at desired target sequences. Dr. Barry Stoddard has studied a family of proteins called meganucleases (also known as homing endonucleases) for decades, seeking to understand how they do their job and what factors affect their activity and stability.

These enzymes seek out and cut specific sequences of DNA, like tiny molecular scissors.  For this reason, they are promising tools for gene editing applications.  In a field dominated by the use of the CRISPR/Cas9, TAL-effector nuclease (TALEN), and zinc-finger nuclease (ZFN) platforms, meganucleases are often overlooked.  Compared to the other gene-editing nucleases available, meganucleases are by far the most challenging to engineer.  However, their small size allows for many more options for delivery into cells, and their high specificity is absolutely necessary for the development of therapeutic or gene-drive reagents.  Therefore, the Stoddard lab continues to research the behaviour of meganucleases and aims to improve the strategies and techniques used to engineer them into custom-specificity enzymes.

Our lab seeks to discover new meganucleases (which exist in nature) and learn as much as possible about them, including how to best engineer them to cut any DNA sequence we desire. For each new meganuclease we identify or engineer, we carry out a variety of experiments designed to learn about the behaviour and characteristics of the enzymes. These analyses include things like how well they cut their DNA target sequence, how specific they are for that exact sequence, and how they perform inside various types of organisms (like bacteria, yeast, or mammalian cell lines). We can also use protein crystallography to determine the three-dimensional structures of the proteins.

As part of the project Target Malaria, the Stoddard lab helps to identify genomic targets towards which to engineer custom-specificity meganucleases for the disruption of fertility in Anopheles mosquitoes.  We then use the tools and techniques developed in our lab to engineer enzymes that will target and cleave the desired fertility gene sequences.