{"doi":"10.1111/pbi.14441","title":"Optimized dCas9 programmable transcriptional activators for plants","abstract":"Understanding how gene expression impacts plant development and physiology is important for crop engineering. Programmable transcriptional activators (PTAs), including CRISPR-Cas activators, have relied on a limited number of transcriptional activation domains (ADs) (Casas-Mollano et al., 2020; Lowder et al., 2018; Pan et al., 2021; Papikian et al., 2019). Usually, the VP64 domain, derived from herpes simplex virus, is fused to a DNA-binding domain to activate target gene expression. In dCas9-based PTAs, binding to a target DNA sequence is afforded by the dCas9-sgRNA ribonucleoprotein. We reasoned there was considerable space for PTA improvement by replacing VP64 with a plant-derived AD. We compiled a list of ADs to test in dCas9-based PTAs in Arabidopsis and Seteria protoplasts as model dicot and monocot species (Figure 1a, Table S1) (Sychla et al., 2022). We started with a literature search of diverse plant-derived transcription factors with known DNA-binding domains. We computationally removed native DNA-binding domains, as these regions are highly conserved and are expected to produce off-target effects if retained in PTA fusions. If an AD was not empirically determined in the literature, we selected motifs enriched in acidic and/or aromatic residues. Acidic and aromatic residue patches are often associated with ADs due to their propensity to form phase separation condensates upon recruitment to a core promoter (Boija et al., 2018). We also added plant-evolved AD sequences from plant pathogen effector proteins such as TALE proteins from Xanthomonas, along with plant-derived sequences from transcription preinitiation complexes such as 14-3-3 scaffolding proteins. The ability of these ADs to function in the context of a dCas9-based PTA was tested in protoplasts from monocot and dicot species (Tables S2 and S3, Figures S1–S7). Specifically, we confirmed that in the Arabidopsis protoplast assay, the SunTag-PTA, in which ADs are recruited to dCas9 using scFv-epitope interactions, outperformed direct translational fusions of ADs to dCas9 (Figure S1). Splitting the genetic components of a dual-luciferase reporter assay across two plasmids decreased the correlated expression of each luciferase reporter that was observed in both Arabidopsis (Figure S2) and Setaria (Figures S3 and S4). AD performance was measured in both the two-plasmid reporter assay in Arabidopsis (Figure S5) and Setaria (Figure 1b) and the one-plasmid assay (Figure S6 for Setaria and Figure S7 for Arabidopsis). Nonspecific activation of the dual-luciferase reporter assay when ADs were expressed without the dCas9 was negligible compared with a positive control (Figure S8). While the specific rank order varied slightly between assays, the ADs DREB2, AvrXa10, DOF1, AtHSFA6b and DREB1 showed transactivation activity comparable or better than VP64 (Figure 1b). We next tested the ability of strong ADs to activate gene expression from endogenous promoters in the Arabidopsis genome (Figure 1c,d) using qRT-PCR. Testing multiple sgRNAs for the FT and CLV3 target genes yielded similar rank-order results, but different fold changes in transactivation (Figure S9). Single guide RNAs targeting the promoters of WUS, CLV3, FT and PAP1 were all capable of driving significant increases in expression. These target genes were selected for their potential use in engineering genetic incompatibility (EGI) in plants. EGI relies on PTA-driven over- or ectopic expression of developmental genes to cause hybrid lethality (Maselko et al., 2020). The largest fold changes in expression were seen for FT, which was overexpressed ten-thousand-fold. The smallest fold changes were seen for PAP1 (10-fold), but this is likely due to the high basal expression level of PAP1 in the absence of a PTA (no-AD control in Figure 1d). Transactivation of both FT and PAP1 yielded expression levels comparable to our housekeeping control, PP2A. Cumulative analysis of different ADs across all four endogenous gene ta","journal":"Plant Biotechnology Journal","year":2024,"id":432390,"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":15,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9565,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","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":989096,"name":"Adam Sychla","orcid":"0000-0002-8262-7557","position":3,"is_corresponding":false},{"id":989097,"name":"Stephen C. Heinsch","orcid":"0000-0003-0406-8734","position":4,"is_corresponding":false},{"id":292378,"name":"Daniel F. Voytas","orcid":"0000-0002-4944-1224","position":5,"is_corresponding":false},{"id":965739,"name":"Michael J. Smanski","orcid":"0000-0002-6029-8326","position":6,"is_corresponding":false},{"id":734914,"name":"Matthew H. Zinselmeier","orcid":"0000-0002-1336-6293","position":0,"is_corresponding":true}],"reference_count":9,"raw_metadata":null,"created_at":"2026-07-19T01:59:35.267142Z","pmid":"39058765","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":[]}