A Transient Assay for Monitoring Zinc Finger Nuclease Activity at Endogenous Plant Gene Targets

Abstract

Advances in plant biology have been frustrated by the lack of an efficient means to create targeted mutations. Zinc finger nucleases (ZFNs) hold much promise for overcoming this limitation: they can be used to generate targeted gene knockouts through imprecise repair of broken chromosomes by non-homologous end joining (NHEJ), or they can stimulate the introduction of specific DNA sequence changes through homologous recombination. Critical to the function of ZFNs is their ability to access and cleave chromosomal target sites. Numerous factors may obscure cleavage, including packaging of DNA into chromatin, DNA methylation, or the presence of other proteins at the target site. Here we describe a transient assay that rapidly assesses ZFN function at chromosomal targets in plant cells. The assay monitors the ability of a ZFN to introduce mutations by imprecise repair through NHEJ, resulting in the loss of a restriction endonuclease recognition sequence. The requirement for the restriction endonuclease recognition sequence coincident with the ZFN spacer region has thus far not been a limiting factor in identifying ZFN target sites in genes of interest suitable for this assay.