megaTALs: a novel rare-cleaving nuclease platform for therapeutic genome engineering

Abstract

Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at "off-target" sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate "megaTAL", in which a TAL effector nuclease is used to "address" a site specific meganuclease adjacent to a single desired target site. This architecture allows the generation of extremely active and hyperspecific compact nucleases that are compatible with all current viral and non-viral cell delivery methods.