Programmable memory as a tool for plant genome engineering

Transkingdom transfer of DNA from Agrobacterium to plants is a stunning biological feat and the basis of the most widely used tool for introducing new DNA into plants. Yet despite decades of use, fundamental gaps remain in our understanding of Agrobacterium-mediated DNA transfer, particularly the dynamics and mechanisms of transferred DNA (T-DNA) integration into the plant genome. These gaps have limited our ability to reliably control transgene insertion. We developed T-SWITCH (T-DNA Sensing With Integrase CHassis), a transgenic Arabidopsis thaliana line that can be used to independently measure T-DNA delivery and integration after Agrobacterium-mediated transformation, to dissect these processes. Using T-SWITCH, we found that most transformation variables, including DNA repair factors, Agrobacterium virulence proteins, promoter choice, and vector architecture, act on T-DNA delivery, not integration. We find that ~17% of T-DNA delivery events do not result in T-DNA integration. Building on this insight, we use T-SWITCH to achieve integration-free genome editing, producing heritable mutations without Cas9 T-DNA in the genome in a single generation.

In a related project we build synthetic genetic circuits that can sense and record specific biological events to achieve greater control over gene expression across the Arabidopsis root system. In Arabidopsis, a single primary root gives rise to secondary (lateral) roots, which subsequently branch into tertiary roots to form an increasingly complex root network over the plant’s life. But because these branch types share the same core regulatory machinery, they have been impossible to control independently. We developed a synthetic genetic “counter” that can record repeated developmental events and enable independent control over gene expression across root types. The system successfully counts to two in heterozygous plants, and we discuss ongoing work to apply it for manipulation of root traits. Together, these systems show how synthetic circuits that record biological events can be used to understand or engineer previously inaccessible plant processes.

Speaker:Jenn Brophy, Assistant Professor of Bioengineering, Stanford University