**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** GENE FLOW FROM GENETICALLY ENGINEERED (GE) CROPS TO SEXUALLY COMPATIBLE WILD OR WEEDY RELATIVES CAN EXACERBATE WEED PROBLEMS WHEN THE CROPS CARRY A FITNESS-ENHANCING TRANSGENE. SEEDS ARE DELIVERABLES OF TRANSGENES. DORMANT SEEDS CAN SURVIVE IN THE SOIL OVER SEASONS, RESULTING IN WEED PERSISTENCE AND THE SPREAD OF AN ESCAPED TRANSGENE IN AGROECOSYSTEMS. THUS, THE GOAL OF THIS PROJECT IS TO DEVELOP A TRANSGENIC MITIGATION STRATEGY BY LINKING A FITNESS-ENHANCING TRANSGENE WITH A CRISPR/CAS9-BASED MULTIPLEX SYSTEM AGAINST SEED-RELATED ADAPTIVE TRAITS TO SUPPRESS SOIL SEED BANKS AND THE SPREAD OF THE TRANSGENE IN CROP-WEED HYBRIDS.THE PROPOSED TRANSGENIC MITIGATION STRATEGY WILL BE TESTED IN WEEDY RICE, THE MOST DIFFICULT TO CONTROL WEED IN RICE-GROWING AREAS, INCLUDING THE SOUTH-CENTRAL UNITED STATES WHERE GENE FLOW FROM CLEARFIELD® RICE HAS BEEN A CONCERN FOR MORE THAN 10 YEARS. SEVERAL GENES ASSOCIATED WITH SEED DORMANCY AND ITS INTERRELATED ADAPTIVE TRAITS HAVE BEEN CLONED FROM BLACK-HULLED WEEDY RED RICE. FUNCTIONAL ALLELES OF THE CLONED GENES HAVE BEEN ELIMINATED FROM CULTIVATED RICE DURING DOMESTICATION. THUS, KNOCKING OUT THE WEED-SPECIFIC GENES HAS NO EFFECT ON CROPS, BUT CAN REDUCE THE FITNESS OF WEEDS AND CROP-WEED HYBRIDS. OBJECTIVES OF THIS PROJECT ARE: 1) TO DEVELOP TRANSGENE-CONTAINING CRISPR/CAS9-BASED MULTIPLEX SYSTEMS FOR HIGH-EFFICIENT MUTAGENESIS OF THE WEED-SPECIFIC GENES IN CULTIVATED RICE, AND 2) TO EVALUATE TRANSGENE-MITIGATING EFFICACY OF THE MULTIPLEX SYSTEMS IN CROP-WEED HYBRID POPULATIONS ACROSS GENERATIONS.THE TRANSGENE BAR FOR HERBICIDE RESISTANCE (HR) AND DIFFERENT SETS OF SMALL GUIDE RNAS (SGRNAS) IDENTIFIED FROM SIX WEED-SPECIFIC GENES HAVE BEEN (OR WILL BE) USED TO DEVELOP TRANSGENE-CONTAINING CRISPR/CAS9-BASED MULTIPLEX SYSTEMS. THE BAR-CONTAINING MULTIPLEX SYSTEMS, LIKE THE BAR::CAS9::SGRNAS TANDEM CONSTRUCT, WILL BE USED TO TRANSFORM THE RICE CULTIVAR NIPPONBARE. RESULTING TRANSGENIC LINES WILL BE ASSAYED FOR HR AND THE COPY NUMBER OF THE TANDEM CONSTRUCT. SINGLE-COPY TRANSGENIC LINES WILL BE EVALUATED FOR TYPES AND FREQUENCIES OF SITE-SPECIFIC MUTATIONS FOR EACH OF THE TARGET GENES. THE TRANSGENIC LINES WITH HIGH-EFFICIENT MUTAGENESIS WILL BE CROSSED WITH ACCESSIONS OF BLACK-HULLED WEEDY RED RICE TO MIMIC TRANSGENE FLOW. THE HYBRID F1S WILL BE EVALUATED FOR SITE-SPECIFIC MUTATIONS AND MUTANT EFFECTS ON SEED TRAITS. THE HYBRID F2 POPULATIONS WILL BE USED TO MAP THE INSERTION LOCUS OF THE TANDEM CONSTRUCT ON THE GENOME, TO EVALUATE MUTANT EFFECTS OF THE MULTIPLEX SYSTEM, AND TO ASSESS THE STRENGTHS OF THE BUILT-IN LINKAGE BETWEEN HR AND EACH OF THE TRAITS. THE F2 SEED SAMPLES WILL BE USED TO MODEL THE LINKAGE (MITIGATING) EFFECT ON GERMINATION DYNAMICS FOR THE TARGET GENES EXPRESSED IN THE EMBRYO TISSUES. F3 FAMILIES WILL BE SELECTED TO MODEL THE LINKAGE EFFECT ON SOIL SEED BANK LONGEVITY FOR THE TARGET GENES EXPRESSED IN THE SEED MATERNAL TISSUES.OUTCOMES FROM THE PROJECT WILL INCLUDE TECHNICAL PARAMETERS AND EFFICACY OF A CRISPR/CAS9-BASED MULTIPLEX SYSTEM ON WEED ADAPTATION, WHICH CAN HELP REGULATORY AGENCIES AND RESEARCHERS BETTER UNDERSTAND CHALLENGES AND PERSPECTIVES OF SYNTHETIC GENE DRIVE IN WEED CONTROL AND TRANSGENIC MITIGATION. THIS PROJECT WILL ALSO PRODUCE NEW GENETIC MATERIALS, SUCH AS THE TRANSGENIC LINES AND THE TRANSGENE-FREE MUTANTS ISOLATED FROM CROP-WEED HYBRID POPULATIONS, AND WILL TRAIN POST GRADUATE RESEARCHERS. THE CRISPR/CAS9-BASED TRANSGENIC MITIGATION STRATEGY TESTED IN RICE COULD BE EXTENDED TO THE OTHER CONSPECIFIC OR CONGENERIC WEED-CROP SYSTEMS.

South Dakota State University, Brookings SD

Investigators