Assembly and characterization of megaTALs for hyperspecific genome engineering applications

Rare-cleaving nucleases have emerged as valuable tools for creating targeted genomic modification for both therapeutic and research applications. MegaTALs are novel monomeric nucleases composed of a site-specific meganuclease cleavage head with additional affinity and specificity provided by a TAL effector DNA binding domain. This fusion product facilitates the transformation of meganucleases into hyperspecific and highly active genome engineering tools that are amenable to multiplexing and compatible with multiple cellular delivery methods. In this chapter, we describe the process of assembling a megaTAL from a meganuclease, as well as a method for characterization of nuclease cleavage activity in vivo using a fluorescence reporter assay.