{"doi":"10.1111/pbi.70096","title":"Towards engineering hybrid incompatibility in plants","abstract":"The potential for gene flow between genetically modified organisms (GMOs) and non-GMO relatives poses a significant challenge to the development and regulatory approval of GMO crops (Wedger et al., 2024), for example, the spread of herbicide resistance transgenes from crops such as rice or sorghum to cross-pollinating weedy species. Addressing this concern, we developed Engineered Genetic Incompatibility (EGI) (Maselko et al., 2017), a system that establishes species-like barriers to gene flow between otherwise sexually compatible populations. EGI employs Programmable Transcriptional Activators (PTAs) to drive lethal over- and/or ectopic expression of tightly regulated genes following undesired hybridization events (Figure 1a,b). A benign mutation of the target promoter in the EGI organism protects it from ill effects of the PTA, which acts as a sentinel for the wild-type (WT) promoter sequence. Given numerous potential PTA targets, multiple mutually incompatible subpopulations are feasible (Maselko et al., 2020). EGI has been demonstrated in yeast as a proof-of-concept (Maselko et al., 2017) and in insects as a strategy for genetic biocontrol of pests (Maselko et al., 2020; Upadhyay et al., 2022). EGI in plants would provide a strategy to halt gene flow between engineered crops and their domestic and wild relatives without altering normal cultivation or propagation practices. Here, we present promising results towards the demonstration of EGI in plants and highlight technical challenges that still need to be overcome. In the model plant, Arabidopsis thaliana, we targeted the WUSCHEL (AtWUS) gene for the development of EGI. AtWUS is crucial for early embryo formation and development. Ectopic, postembryonic AtWUS expression induces aberrant somatic embryogenesis, resulting in callus formation or post-germination growth arrest and death (Zuo et al., 2002). Prior work has shown that single guide RNAs (sgRNAs) targeting PTAs to regions upstream of the transcriptional start sites (TSS) trigger strong transcriptional activation (Casas-Mollano et al., 2023). Thus, we targeted four sites within 500 bp of the TSS in AtWUS (Figure 1c) for activation with the SunTag PTA (Papikian et al., 2019). SunTag utilizes activation domain (AD) scaffolding, where a VP64 AD is fused to a scFv antibody while a multimeric GCN4 epitope tail is fused to dCas9. Each scFv antibody recognizes a GCN4 epitope, recruiting multiple ADs to a single dCas9 molecule (Papikian et al., 2019). We crossed a PTA expressing line with a line expressing 4 sgRNAs targeting the AtWUS promoter. The expected ectopic embryo formation in the F1 progeny was observed (Figure S1), giving us confidence to pursue EGI using WUS overexpression. Individual sgRNAs were expressed in Arabidopsis protoplasts with sgRNA1 showing the strongest AtWUS activation (Figure 1c), guiding subsequent EGI construction. We then mutated the AtWUS promoter to block PTA binding in the EGI plant. A vector (Figure S2) expressing a catalytically active Cas9, sgRNA1 and the RFP-based FAST marker gene (Shimada et al., 2010) for Cas9 selection was used to transform Arabidopsis plants. Screening RFP-positive T1 plants confirmed multiple unique fixed biallelic AtWUS promoter indels across 30 T1 lines. After segregating out Cas9 in the T2 generation (RFP-negative plants), we advanced two lines for EGI prototyping that have homozygous AtWUS promoter indels (Figures 1d and S3) and no apparent phenotype. We transformed these lines with a T-DNA containing the PTA, sgRNA1 targeting the WT AtWUS promoter and FAST marker (Figure S4). Evidence of successful transformation included accumulation of RFP in seeds (Figure S5). This EGI system uses MoonTag PTAs, which we have found to outperform SunTag PTAs in transgenic plants (Casas-Mollano et al., 2023; Zinselmeier et al., 2024). After floral dip transformation, RFP+ T1 seeds were germinated in soil, showing no obvious phenotype, suggesting that the promoter mutations success","journal":"Plant Biotechnology Journal","year":2025,"id":533572,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9525,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":989095,"name":"J. Armando Casas-Mollano","orcid":"0000-0001-7246-7142","position":1,"is_corresponding":false},{"id":989698,"name":"Jonathan Cors","orcid":null,"position":2,"is_corresponding":false},{"id":1344694,"name":"Sávio Siqueira Ferreira","orcid":"0000-0001-9926-9066","position":3,"is_corresponding":false},{"id":292378,"name":"Daniel F. Voytas","orcid":"0000-0002-4944-1224","position":4,"is_corresponding":false},{"id":965739,"name":"Michael J. Smanski","orcid":"0000-0002-6029-8326","position":5,"is_corresponding":false},{"id":734914,"name":"Matthew H. Zinselmeier","orcid":"0000-0002-1336-6293","position":0,"is_corresponding":true}],"reference_count":13,"raw_metadata":null,"created_at":"2026-07-19T02:51:32.301795Z","pmid":"40263770","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}