Silencing Seed Dormancy Genes to Mitigate Risk of Transgene Flow to Weedy Rice

The flow of fitness-enhancing transgenes from genetically modified crops into wild/weed relatives may cause serious ecological and economic consequences. Seed dormancy (SD) is a key adaptive trait that distributes germination over time, resulting in weed persistence in agroecosystems. Thus, silencing major genes controlling SD would reduce the adaptive fitness of weeds. SD-enhancing genes cloned from weedy rice include SD7-1, SD7-2, SD12a, SD12b, and SD12c. The goal of this study was to develop a transgenic mitigation (TM) strategy using SD gene-silencing structures as mitigating factors to reduce the risk of transgene flow to wild/weed populations. TM vector constructs consisted of the Bar herbicide resistance (HR) transgene linked to either an RNA interference (RNAi) or CRISPR/Cas9 SD gene-silencing cassettes. In the RNAimediated TM strategy, a two-locus and a three-locus TM constructs were designed to target two or three SD genes in weedy rice, respectively. Hemizygous T0 plants were crossed with the weedy rice line Ludao to generate HR and herbicide susceptible (HS; without TM construct) genotypes for fitness evaluations under greenhouse and field conditions. The two-locus TM construct significantly reduced (p \u3c 0.0001) the degree of dormancy among HR genotypes when compared to the HS genotypes. However, the three-locus TM construct could not reduce the degree of dormancy in HR genotypes when evaluated under greenhouse and field conditions. To maximize silencing efficiency, a CRISPR/Cas9-based TM construct was designed to knockout six SD genes in weedy rice simultaneously. Of the mutations identified in the T0 plants, 62% were deletions, 33% insertions and 5% substitutions, and were classified into homozygous, heterozygous and biallelic types. The Cas9-induced mutations were found to be heritable when a biallelic T0 plant was crossed with the weedy rice line SS18-2 to generate F1 hybrid plants, but no new mutations were observed in the SS18-2 allele for three of the six targeted genes. The results obtained in this study proved that a TM strategy based on SD gene-silencing mitigating factors is feasible. However, the tandemly linked mitigating factors need to be modified to enhance their silencing efficiency -- a CRISPR/Cas9 gene drive approach was thus proposed