Postdoctoral Research Associate Oregon State University
Stable insertion of gene editing components such as CRISPR/Cas9 through Agrobacterium-mediated transformation is the most common method for gene editing in plants. For most crop species, the editing machinery is removed by sexual segregation leaving an edited trait and the transgene removed. For asexually propagated plants, such as many forest tree species, this process is not feasible. To overcome this obstacle, we sought to build a system where gene editing components could be removed somatically during transformation and regeneration. This method builds on previous gene excision methodologies using site-specific recombinases. Due to the asynchronous development characteristic of organogenic regeneration, we expected an excision scheme induced within meristematic cells in shoot primordia could be better suited to this process compared with inducible systems. To find suitable promoters to drive recombinase expression in hybrid poplar emerging shoots, we analyzed transcriptional fluorescent reporter lines using promoters of characterized Arabidopsis shoot meristem genes and their homologs from Populus trichocarpa. Promoters of the WUSCHEL (WUS), SHOOT MERISTEMLESS (STM), and COLD SHOCK PROTEIN 3 (CSP3) genes provided adequate expression in emerging shoot primordia during regeneration to warrant further analysis. We modified an existing gene editing vector system to perform developmental transgene excision by adding recombinase recognition sites, fluorescent reporters to observe insertion and excision, and CRE recombinase fused to the glucocorticoid receptor for increased control of CRE activity. We will present our initial editing and excision rates obtained with the pCSP3 driven recombinase construct, using PHYTOENE DESATURASE (PDS) knockouts to determine editing rates. We propose this system could be an effective method for the routine somatic removal of gene editing components in asexually propagated plants, for further line development and regulatory approval.
Coauthors: Cathleen Ma – Oregon State University;Steven Strauss – Oregon State University