Harnessing Double Strand Break Repair for Targeted Mutagenesis

Methods for precisely altering DNA sequences in living cells enable detailed functional analysis of genes and genetic pathways.  Targeted genome modification also has many real-world applications, ranging from improving crop plants to treating genetic disorders in people.  Our group has enabled efficient targeted genome modification through the use of sequence-specific nucleases, which are engineered to create chromosome breaks in target genes of interest.  The cell’s DNA repair pathways are then harnessed to effect desired sequence modifications at or near the break site.  As a founding member of the Zinc Finger Consortium, we have worked collaboratively to develop efficient methods for the design of zinc finger nucleases (ZFNs) that recognize unique genomic targets with high specificity and affinity.  Using our ZFNs, we have demonstrated targeted gene modification in plants (tobacco) at frequencies exceeding 10% of transformed cells.  We have also created targeted gene knockouts in Arabidopsis and soybean, thereby enabling the recovery of plants with mutations in genes of interest. More recently, we have worked collaboratively to develop methods to engineer the DNA binding domain of Transcription Activator-Like (TAL) effectors.  When fused to FokI nuclease, the TAL effector DNA binding domain creates chromosomal breaks at specific DNA sites.  Reagents and protocols for the rapid assembly and testing of custom TAL Effector Nucleases (TALENs) have been developed, and we and others have used TALENs to create mutations in human, Drosophila, C. elegans, zebrafish and Arabidopsis genes.