Genome Engineering

Targeted genome editing is dependent on our ability to induce a DNA double strand break (DSB) at a specific location in the genome and on the mode of repair.  For example, repair by non-homologous end joining is error-prone in plants and can lead to mutagenesis, usually seen as small indels.  Repair via homologous recombination can lead to targeted gene replacement (GT) if the homologous DSB repair template originates from a delivered exogenous DNA or to targeted gene conversion or crossing over when repair is done using an homologous chromosome.  Developing these technologies has been an outcome of our interest in DSB repair.  The recent use of custom designed nucleases, ZFNs, TALENs and in particular CRISPR-Cas is opening the prospect for both targeted mutagenesis in plants.  In a comparative study we have shown that CRISPR-Cas is the most promising technique due to its ease of design and high efficiency. 
Targeted knockout:  We have shown in tomato that CRISPR-Cas is highly effective in targeted gene knockout generating a high rate of mutations already at the early developmental stages of the plant. It generates some preferential repair footprints that are dependent on the site of breakage.
Targeted crossover:  We are taking advantage of the fact that some breaks are repaired by homologous recombination, in order to induce targeted crossover between homologous chromosomes.
Gene targeting: We have shown that proteins such as RAD54 or AtRAD52 overexpression can enhance the rate of gene targeting but only to a limited extent.  We are currently using viral vectors combined with CRISPR-Cas DSB induction to develop efficient gene targeting in plants.
T-DNA integration: A powerful tool for genome engineering is transformation using the Agrobacterium-derived T-DNA vector. It is still unclear whether T-DNA integration is a random event or is biased towards particular genomic regions.  The kinetics of integration is also still unclear. Using High throughput sequencing, we showed in collaboration with Prof. Theodore Muth from Brooklyn College, that the T-DNA can integrate as soon as 6hrs after infection.  In addition, we show that there is a great difference between selected and unselected transformation events.  Selected events are mostly in gene-rich regions, distal from the centromere and in hypomethylated and nucleosome-free regions.  Unselected events occur throughout the chromosome and including in heterochromatin regions.