The generation of a double-strand break led to error-prone non-homologous end joining (NHEJ), resulting in frequent mutations of KMT2D. Using this pair of ZFNs to mutate KMT2D in medulloblastoma cell lines, we introduced nucleotide alterations in one allele of KMT2D through a single round of transient nuclease expression. Here, KMT2D is linked to medulloblastoma.