Objective
The main objective of the project is to develop a gene drive system for the suppression of Kochia and waterhemp.
(1) Identification, selection, and validation of reproductive genes via a transcriptomic analysis of male vs. female floral tissues in Kochia and waterhemp.
(2) Development of a CRISPRCas9 construct for gene drive (including gRNA + rescue gene + selectable marker + promoter) and functional validation in Kochia and waterhemp.
(3) Kochia and waterhemp transformation via the Agrobacterium-mediated method, and screening and validation of gene drive in both weeds.
Project Description
Kochia scoparia and waterhemp are among the most economically damaging, herbicide-resistant weed species in North America, including the Canadian Prairies. In canola-growing regions, kochia is particularly aggressive due to its high fecundity (10,000+ seeds per plant), wind-dispersed seeds (tumbling), and short life cycle. It is monoecious and self-compatible, characteristics that make it both invasive and difficult to manage through cultural and chemical means. Its seedbank only persists for 1-2 years, however, meaning that short-term management strategies can effectively deplete local populations. Kochia has evolved resistance to multiple herbicide classes, including glyphosate (Group 9), ALS inhibitors (Group 2), synthetic auxins (Group 4), and PPO inhibitors (Group 14), with resistance often stacked within populations (Stahlman, 2016). This plant appears to be spreading within the Prairies, with in-field populations being reported further north than the ‘typical’ kochia distribution of the past. Waterhemp has emerged as a global model for herbicide resistance evolution, now exhibiting resistance to seven herbicide site-of-action groups, including both target-site and non-target-site (NTS) metabolic resistance mechanisms. While early resistance in waterhemp was primarily target-site-based, new resistance mechanisms emerging after 2013 are predominantly NTS. The widespread prevalence of NTS resistance highlights the urgent need for sustainable management strategies for this weed. Waterhemp has become an issue in Manitoba in particular. It has been found through all U.S. states that border Saskatchewan and Alberta as well, indicating it is likely a matter of time before it is found across the Prairies. This species typically arrives in new locations already resistant to many herbicides, amongst which glyphosate resistance is common. These trends point to an escalating crisis in modern agriculture, with Kochia and other Amaranthus spp. causing important yield losses and threatening sustainable crop production. Therefore, there is a critical need for innovative, non-chemical weed management solutions to protect canola productivity and reduce herbicide dependency. While herbicide-resistant weeds like Kochia and waterhemp continue to evolve, genomics-based approaches can offer new insights into herbicide resistance and the biology of these species, allowing the development of novel, non-chemical control strategies. Understanding the molecular mechanisms behind herbicide resistance, how these mechanisms confer cross-resistance to other herbicides, and how resistance evolves remains a critical research priority. Transcriptomic and genomic approaches are already helping to elucidate these mechanisms, and the results from these studies will inform more effective resistance-mitigation strategies. One promising avenue is the application of gene drive systems, which offer a biology-based approach to suppressing invasive weed populations by biasing the inheritance of specific genetic traits. This way, it is possible to increase the frequency of lethal alleles in weed populations, leading to sterile plants or abortive seeds. These gene-drive technologies, particularly CRISPR/Cas9-based systems, are particularly well-suited for highly outcrossing species like waterhemp. Gene drives are already being explored for invasive plant management, including in species like common tansy, where gene drives targeting female fertility are proposed to suppress seed production and limit population spread. One promising approach is the Cleave and Rescue (ClvR) system, which uses CRISPR technology to disrupt essential reproductive genes, such as those involved in flower development or gametogenesis. The ClvR system simultaneously introduces a rescue gene that ensures only drive-carrying individuals survive and reproduce. Both Kochia and waterhemp are strong candidates for gene drive applications because of their biological and genetic traits. ClvR-based systems could significantly reduce seed production over multiple generations, ultimately suppressing population density. These gene drive approaches rely on CRISPR/Cas9 systems that cut the genome at specific sequences and drive DNA repair through homologous recombination, rather than the non-homologous end-joining pathway, to ensure successful drive propagation. High rates of homologous repair are critical for the efficiency of gene drives in weeds like Kochia and waterhemp. Previous gene drive efforts have demonstrated success in mosquitoes, with over 97% of genome edits propagated, but similar efforts in other species like fruit flies have been less successful (< 78%). Achieving high rates of homologous repair in Kochia and waterhemp will be a necessary step in demonstrating the viability of gene drive systems for weed management.
