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We demonstrate two synthetic single-cell systems that can be used to better understand how the acquisition of an orphan gene can affect complex phenotypes. The Arabidopsis orphan gene,Qua-Quine Starch(QQS) has been identified as a regulator of carbon (C) and nitrogen (N) partitioning across multiple plant species.QQSmodulates this important biotechnological trait by replacing NF-YB (Nuclear Factor Y, subunit B) in its interaction with NF-YC. In this study, we expand on these prior findings by developingChlamydomonas reinhardtiiandSaccharomyces cerevisiaestrains, to refactor the functional interactions between QQS and NF-Y subunits to affect modulations in C and N allocation. Expression ofQQSinC. reinhardtiimodulates C (i.e., starch) and N (i.e., protein) allocation by affecting interactions between NF-YC and NF-YB subunits. Studies inS. cerevisiaerevealed similar functional interactions between QQS and the NF-YC homolog (HAP5), modulating C (i.e., glycogen) and N (i.e., protein) allocation. However, inS. cerevisiaeboth the NF-YA (HAP2) and NF-YB (HAP3) homologs appear to have redundant functions to enable QQS and HAP5 to affect C and N allocation. The genetically tractable systems that developed herein exhibit the plasticity to modulate highly complex phenotypes. 

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.