Transient Testing of Enzymes Designed for Genome Editing in Maize
Successful genome editing is associated with the ability to generate double strand breaks (DSB) efficiently at specific chromosomal locations. Recently, enzymes called TALENs (transcription activator like effector nucleases) have been tested for this purpose. TALENs consist of a DNA binding domain and a DNA cleaving or nuclease domain. They function as dimers; two TALEN proteins interact together to produce a DSB in the DNA. The efficiency of TALENs is influenced by the ability to access and bind the target site and of the two TALENs to dimerize. We have assembled a transient assay system in maize to test which TALENs are most effective at generating DSBs. We generated a modified fluorescent protein reporter gene that contains the DNA binding site for the TALENs. When this gene is expressed following DNA bombardment into maize embryos, it is non-functional and no fluorescence is observed. Genes encoding TALENs are then co-bombarded with the reporter gene plasmid. When DSBs occur, DNA repair machinery of the maize cells will repair the reporter gene generating a functional fluorescent protein. The number of fluorescent cells recorded is a measure of TALEN activity. Results from several TALENs and other DSB enzymes, such as RNA-guided cas9 will be discussed.