{"doi":"10.1093/pcp/pcae117","title":"Letter to the Editor: Gene Targeting in <i>Arabidopsis</i> through One-Armed Homology-Directed Repair","abstract":"Gene targeting (GT) refers to the precise modification of genomic sequences by repairing DNA breaks through homology-directed repair (HDR). GT offers many advantages over traditional T-DNA transgenics such as seamless insertion of cargo without residual selection markers, but it has been challenging to perform GT in plants. Targeted insertions in Arabidopsis have been reported (Miki et al. 2018, Wolter et al. 2018, Peng et al. 2020, Zhang et al. 2022), but they typically rely on sequential transformation into a background already expressing Cas9, and insertion site selection is limited by the available PAM sites in the desired region. We generated targeted insertion lines using a slightly modified version of the sequential transformation system reported previously (Supplementary Fig. S1). In genotyping our insertion lines, we found that most lines had detectable HDR events on only a single side, suggesting that one-sided invasion may have occurred (Supplementary Fig. S2). Moreover, we found that the selection marker often segregated with the insertion in subsequent generations, suggesting that the extra T-DNA material was linked to the insertion site. We performed whole-genome sequencing (WGS) on several of these lines and found a variety of unexpected repair events, including tandem T-DNA and donor insertions (Supplementary Fig. S2). In addition, some lines had large-scale genome alterations, including chromosomal inversions and translocations (Supplementary Figs. S2, S3). In some cases, we retransformed these lines with CRISPR/Cas9 constructs containing gRNAs to target and remove the extra sequences (Supplementary Fig. S4.) Our results from these initial GT studies underscore the independence of each side of a double-strand break (DSB) in terms of their repair process and the low probability of achieving HDR on both free ends. The probability of a perfect insertion is made even lower by the fact that for ideal GT, both ends of the DSB should repair using the same donor template. Thus, in order to increase the fidelity of our GT system, we looked to uncouple the two repair events such that successful GT did not rely on both sides of the DSB hybridizing with a shared donor. We developed an improved technique for GT in Arabidopsis based on the one-armed HDR (oaHDR)/single homology arm donor-mediated intron-targeting integration (SATI) technique reported in animal systems (Suzuki et al. 2019), (Fig. 1A). Working in mice, Suzuki et al. demonstrated that targeted insertions were possible when the homology regions only matched to a single side of the DSB. By the oaHDR method, only one side of the DSB must be repaired by HDR, and the other is free to repair via non-homologous end joining (NHEJ). NHEJ typically results in short insertions or deletions, but the authors mitigated any negative consequences of NHEJ by designing gRNAs that did not induce DSBs within coding sequences. We applied the SATI technique to Arabidopsis and discovered that oaHDR is a feasible method for precisely inserting cargo sequences into genes (Fig. 1). By designing gRNAs that target introns or intergenic sequences, small indel ‘scars’ leave no predicted effect on gene function. This system has the additional benefit of permitting a wide range of guide RNA selections. An oaHDR-based GT system for Arabidopsis. (A) A schematic of the GT system and possible repair methods. The GT construct encodes two separate T-DNA units, bound on either side by left and right borders (LB and RB). The first T-DNA contains the donor sequence, which has the cargo (green) flanked by two homology sequences (orange, H1 and H2). The second T-DNA contains the gRNA, which targets the cut site, the coding sequence for Cas9, and a selection marker. After Agrobacterium infiltration, the Cas9 and gRNA are expressed in plant cells and the Cas9–gRNA complex generates a DSB at the target site, which is designed to be in a non-coding region. One end of the DSB repairs through oaHDR using the ","journal":"Plant and Cell Physiology","year":2024,"id":484589,"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":9,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9617,"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":616067,"name":"Bradley W. Abramson","orcid":"0000-0003-0888-3648","position":1,"is_corresponding":false},{"id":458490,"name":"Xinhua Dai","orcid":"0000-0002-8149-5513","position":2,"is_corresponding":false},{"id":1327227,"name":"Ruofan Kang","orcid":null,"position":3,"is_corresponding":false},{"id":1327228,"name":"Ethan Young","orcid":null,"position":4,"is_corresponding":false},{"id":28822,"name":"Todd P. Michael","orcid":"0000-0001-6272-2875","position":5,"is_corresponding":false},{"id":232351,"name":"Yunde Zhao","orcid":"0000-0002-7224-8449","position":6,"is_corresponding":false},{"id":727097,"name":"Michael Mudgett","orcid":"0000-0001-9585-8082","position":0,"is_corresponding":true}],"reference_count":10,"raw_metadata":null,"created_at":"2026-07-19T02:07:42.971417Z","pmid":"39366925","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":[]}