Learning the grammar of plant regulatory DNA for targeted engineering of gene expression

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Engineering of regulatory regions in plant genomes has the potential to facilitate rapid crop improvements necessary for ensuring food security. This project will analyze plant cis-regulatory elements to determine the ways in which they interact—the regulatory grammar—and build tools to engineer regulatory DNA for crop improvement. Gene expression is controlled by cis-regulatory elements that include promoters, enhancers, silencers and insulators. Engineering such elements is a promising strategy to improve crop traits. Changes in regulatory regions often cause tissue- or condition-specific changes in gene expression, and hence can be less deleterious than coding changes. Importantly, evolution and domestication both have frequently acted on regulatory DNA. However, we still lack sufficient knowledge of cis-regulatory elements and their grammar to make routine the engineering of these elements for crop improvement. I recently developed plant STARR-seq, a massively parallel reporter assay that can characterize the activity of cis-regulatory elements. In this project, I will use plant STARR-seq to further our understanding of plant gene regulation: (1) I will characterize plant insulators and silencers, as these elements are needed to tightly control the expression of transgenes for effective crop engineering; (2) I will study the interactions within and between cis-regulatory elements; and (3) I will study how higher-order interactions between cis-regulatory elements and trans-acting proteins fine-tune plant gene expression. These studies will lead to a deeper understanding of the rules that govern the activity and strength of plant regulatory DNA and will enable us to accurately predict and manipulate plant gene expression. In parallel to studying plant regulatory grammar, I will establish a toolkit for transgene-free genome editing of cis-regulatory elements. Current technical solutions for targeted engineering of plant genomes face public skepticism about transgenic organisms and concerns around off-target effects. In this project, I will develop an alternative approach to plant genome engineering by using editing proteins that are expressed in Agrobacteria and subsequently transferred to plant cells without a concomitant transfer of DNA. This system will yield transgene-free plants and reduce the number of off-target mutations since the editing proteins will not be produced constitutively.