Background: Upland cotton (Gossypium hirsutum) is a major natural fiber crop and an important model for studying genome evolution and gene function in polyploid plants. However, its large and highly redundant genome presents substantial challenges for efficient and coordinated multiplex genome editing. Methods: Here, we developed a high-efficiency CRISPR/Cas12a-based multiplex genome editing system in cotton by integrating a tRNA–crRNA polycistronic expression strategy with a Bean yellow dwarf virus (BeYDV)-derived replicon. Results: This platform enabled coordinated expression of multiple crRNAs and simultaneous targeting of 16 loci within a centromere-proximal region of chromosome D03 (18.65–24.47 Mb). In individual transgenic lines, up to 10 target sites were edited concurrently, with nine targets exhibiting editing efficiencies above 56% and the highest efficiency reaching 96.46%. High-density multiplex editing predominantly induced small insertions and deletions at target loci. Notably, edited plants exhibited reduced growth and pronounced cytological abnormalities, including chromosome bridges, lagging chromosomes, and abnormal meiotic products. Transcriptome analysis revealed widespread dysregulation of genes involved in chromosome segregation and cell cycle regulation. Despite these functional perturbations, HiFi long-read sequencing detected no large-scale chromosomal rearrangements, indicating that genome instability arises from cumulative local perturbations rather than global structural alterations. Conclusions: Together, our results establish an efficient multiplex genome editing platform in cotton and highlight potential constraints of high-density editing on genome stability in complex plant genomes.

Purpose: Gibberellins (GAs) are key phytohormones that regulate plant growth, development, and responses to environmental stress, and their metabolism is mediated by 2-oxoglutarate-dependent dioxygenases (2OGDs). Cotton (Gossypium spp.) is a polyploid crop with a complex genome; however, the evolutionary characteristics and stress-responsive regulation of GA-related 2OGDs remain poorly understood. This study aimed to systematically investigate the evolution, expression patterns, and stress-associated regulation of the cotton 2OGD multigene family, with particular emphasis on GA-related members. Methods: 2OGD genes were identified genome-wide in four Gossypium species and Arabidopsis thaliana. Phylogenetic relationships, gene structures, conserved motifs, cis-acting regulatory elements, and synteny were analyzed. Transcriptomic data from multiple tissues and developmental stages, together with time-course RNA-seq under salt stress, were examined. Transcriptome–metabolome association analysis, endogenous GA quantification, and predicted protein–protein interaction analysis were conducted. Results: A total of 583 2OGD genes were identified and classified into three major classes, including a Class C group comprising GA2ox, GA3ox, and GA20ox genes. Polyploidization-associated duplication contributed to the expansion of the 2OGD family, and most duplicated gene pairs exhibited signatures of purifying selection. GA-related 2OGDs displayed conserved motif compositions with variation in cis-acting elements. Promoter analysis identified abundant hormone-responsive, stress-responsive, and growth-related cis-elements, suggesting complex regulatory control of GA-related 2OGDs in cotton. Under salt stress, GhGA2OX1 and GhGA20OX2 were upregulated, whereas GhGA3OX1 was downregulated, accompanied by reduced endogenous GA levels. Conclusions: GA-related 2OGDs in cotton are transcriptionally responsive to salt stress and are associated with changes in GA metabolism, providing a basis for future functional studies.