Defining off-target cleavage in a pair of Zinc Finger Nucleases
Poster Apr 13, 2012
K. Mukherjee, D. Carroll
Studies on Zinc Finger Nucleases (ZFN) have shown that they can be toxic in organisms. This is potentially due to ZFN cleavage at multiple off-target sites. In applications of ZFNs in human gene therapy, this off-target cleavage is intolerable. We are attempting to develop a procedure to identify these off-target sites in Drosophila. We can then analyze every new ZFN pair for potential off-target cleavage and select and redesign it to work more efficiently.
Multiplexing cell-based assays is possible using 3D culture models that are larger and more complex than monolayers
Real-time detection methods to measure live or dead cells provide much flexibility for multiplexing
All multiplexed assay combinations should be verified using appropriate controls for each 3D cell culture model.