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Summary Genome editing is a revolution in biotechnology for crop improvement with the final product lacking transgenes. However, most derived traits have been generated through edits that create gene knockouts.Our study pioneers a novel approach, utilizing gene editing to enhance gene expression by eliminating transcriptional repressor binding motifs.Building upon our prior research demonstrating the protein‐boosting effects of the transcription factor NF‐YC4, we identified conserved motifs targeted by RAV and WRKY repressors in theNF‐YC4promoters from rice (Oryza sativa) and soybean (Glycine max). Leveraging CRISPR/Cas9 technology, we deleted these motifs, resulting in reduced repressor binding and increasedNF‐YC4expression. This strategy led to increased protein content and reduced carbohydrate levels in the edited rice and soybean plants, with rice exhibiting up to a 68% increase in leaf protein and a 17% increase in seed protein, and soybean showing up to a 25% increase in leaf protein and an 11% increase in seed protein.Our findings provide a blueprint for enhancing gene expression through precise genomic deletions in noncoding sequences, promising improved agricultural productivity and nutritional quality. 

Abstract Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T‐DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site‐specific recombinases. Finally, we use a series of dual‐luciferase assays to measure contributions to expression from promoters, terminators, and from cross‐cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.