Genome engineering using DNA-binding proteins: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)

Abstract

<p>Over the past two decades, research groups in both academia and private industry have developed key technologies, including viral delivery vectors and engineered transposon-based or zinc finger protein-based nucleases, towards achieving the long-sought goal of therapeutic genome editing in humans. To date, Zinc Finger Nucleases (ZFNs) have been the most promising reagents for potential therapeutic applications in humans, but the recently characterized Transcription Activator Like Effector (TALE) proteins may soon change this status quo. Although it remains to be seen whether nucleases based on these proteins (TALENs) will be as broadly applicable and effective as ZFNs, based on initial reports, TALENs look very promising. Currently, the primary advantage of TALENs is that the DNA binding code for TALENs appears to be simple and robust, making their synthesis relatively simple.</p> <p>In this dissertation, I summarize advances made in the field of genome editing over the past decade and compare and contrast the currently available tools, focusing on ZFNs and TALENs. Specifically, I describe our efforts to make ZFN technology more accessible by designing and implementing models to help researchers choose target sites that are most amenable to targeting using ZFNs. Also, to help explore the potential of TALENs as tools for genome editing, I describe the development of a simple protocol to aid in constructing TALENs. As ZFNs become easier to use, and TALENs become more robust, the use of genome editing techniques as therapeutics appears poised to become reality in the near future.</p>