<scp>CRISPR</scp> /Cas9‐Mediated Specific Knockout of <scp> <i>GhCesA4</i> /7/8 </scp> Improves Cotton Fibre Quality

Upland cotton (Gossypium hirsutum), a globally cultivated crop of significant importance to the textile industry, undergoes five developmental stages: initiation, elongation, transition, secondary cell wall (SCW) synthesis and maturation (Haigler et al. 2012). The stages from elongation to SCW synthesis determine three key fibre traits: length, strength and fineness—the latter two being influenced by SCW thickness. However, fibre length and SCW thickness exhibit an inverse relationship in practice. Therefore, concurrently optimising these conflicting traits to cultivate cotton fibres with enhanced length, strength and fineness persists as a key challenge in modern breeding. Cellulose, synthesised by cellulose synthase (CesA), constitutes up to 95% of the dry weight of mature cotton fibre. The upregulation of GhCesA genes specifically involved in SCW biosynthesis (referred to as SCW-GhCesAs) occurs during the late stage of fibre elongation and coincides with the initiation of SCW deposition (Qiao et al. 2025). To elucidate their functional roles and potential for fibre quality improvement, we employed CRISPR/Cas9 to generate targeted knockout mutations of SCW-GhCesAs, aiming to improve fibre length, strength and fineness. The allotetraploid Gossypium hirsutum genome contains 12 SCW-GhCesAs genes, categorised into the GhCesA4, GhCesA7 and GhCesA8 subfamilies, each represented by four homologous copies (Table S1) (Zhao et al. 2022). Most of these genes showed fibre-predominant expression, except for GhCesA8-1At and GhCesA8-1Dt, which contain a 150–200 amino acid deletion in their N-terminus. Therefore, the remaining 10 SCW-GhCesAs were selected for further study (Figure 1a; Figures S1 and S2). Transcriptome profiling throughout fibre development demonstrated that SCW-GhCesAs were upregulated beginning at the late elongation stage and maintained high expression thereafter (Figure 1b). Furthermore, correlation analysis across 251 cotton accessions revealed that their expression was negatively correlated with fibre length but positively correlated with micronaire—an indicator of cell wall thickness (Figure 1c). Since conservative knockout of all copies of any gene among GhCesA4/7/8 results in nearly complete loss of cellulose deposition (Wen et al. 2022), we adopted a nonconservative gene editing strategy. For GhCesA4 and GhCesA7, a single gRNA targeting conserved sequences in both At and Dt subgenomes was used to simultaneously disrupt their homologous copies: GhCesA4-1At/Dt, GhCesA4-2At/Dt, GhCesA7-1At/Dt and GhCesA7-2At/Dt. For GhCesA8, specific gRNAs were designed to target GhCesA8-2At or GhCesA8-2Dt (Table S2) individually. Agrobacterium-mediated transformation in cotton generated multiple SCW-Ghcesas mutant lines, from which six with high editing efficiency—Ghcesa4-1At/Dt#2, Ghcesa4-2At/Dt#3, Ghcesa7-1At/Dt#4, Ghcesa7-2At/Dt#5, Ghcesa8-2At#3 and Ghcesa8-2Dt#2—were selected for further investigation (Figure S3; Data S1, S2). Phenotypic analysis demonstrated that, compared to WT, the SCW-Ghcesas mutants exhibited longer fibre throughout elongation, transition, SCW synthesis and mature stages (Figure 1d,e). Accordingly, TEM revealed a significant reduction in cell wall thickness in the mutants (Figure 1f,g). This decrease was initially observed during the SCW synthesis stage and persisted into maturity (Figure S4). Moreover, quantitative analysis of crystalline cellulose content in mature fibres showed that all SCW-Ghcesas mutants had significantly lower levels than WT (Figure 1h). Although cellulose accumulation and Calcofluor White fluorescence increased progressively during development in both WT and mutants, fluorescence intensity remained markedly lower in the mutants at all stages examined (Figure 1i,j; Figure S5). Multigeneration fibre quality evaluation further demonstrated that SCW-Ghcesas mutants exhibited significant improvements in key fibre quality parameters compared to WT (Figure 1k–m). Using T4 generation data as a representative example, SCW-Ghcesas mutants displayed elongated fibres compared to the WT. Notably, the Ghcesa8-2At mutant exhibited the most pronounced increase in fibre length (28.66 ± 0.23 mm), representing a 4.31%–6.22% improvement over WT (27.16 ± 0.59 mm). In contrast, micronaire values were substantially reduced in mutants, with Ghcesa7-2At/Dt showing the strongest decline (3.59 ± 1.09), equivalent to a 35.41%–37.37% decrease compared to WT (5.62 ± 0.09). Furthermore, fibre strength in SCW-Ghcesas mutants was significantly improved, with Ghcesa7-1At/Dt (29.47 ± 0.31) achieving an 11.75%–14.43% increase over the WT (26.13 ± 0.41) (Figure 1m). Taken together, the nonconservative knockout of SCW-GhCesAs improves key parameters of cotton fibre quality—resulting in longer, finer and stronger fibres. Notably, interannual variation in fibre phenotypes was observed. Compared to the T2 and T4 generations, both T3-generation mutants and WT plants showed significantly reduced fibre length and strength, with limited quality improvement in mutants (Figure 1k–m). This variation was concurrent with temperature anomalies during critical fibre development stages in the T3-generation (Figure S6), supporting a mechanism whereby temperature alters CesA motility, thereby modulating cellulose synthesis efficiency (Fujita et al. 2011). Another key finding was that the Ghcesa7-2At/Dt mutant, which had the most pronounced reduction in cellulose content, consistently exhibited the lowest micronaire values (Figure 1h,k–m). Given the high expression abundance of GhCesA7-2At and GhCesA7-2Dt among GhCesAs (Figure S7), we analysed correlations between SCW-GhCesA expression and fibre quality parameters in SCW-Ghcesas mutants. Significant negative correlations were found between SCW-GhCesA expression and both micronaire and cell wall thickness (Figure 1n,o; Figures S8 and S9). These data indicate that variation in SCW-GhCesAs expression levels leads to differences in fibre quality among the SCW-Ghcesas mutants. In summary, CRISPR/Cas9-mediated nonconservative knockout of SCW-GhCesAs led to cotton with longer, stronger and finer fibre, among which the Ghcesa7-2At/Dt mutant exhibited the most pronounced improvements. These results demonstrate that precise editing of SCW-GhCesAs offers a promising strategy to overcome the long-standing trade-off between fibre length and thickness. L.T., X.Z. and S.J. designed and guided the project. L.Q. performed experiments and drafted the manuscript. H.T. helped perform the correlation analysis. Z.Y. offered experimental technical assistance. This work was supported by funding from the National Natural Science Foundation of China (Project No. 32172071), the National Key R&D Program of China (Project No. 2022YFF1001400) and Biological Breeding-Major Projects in National Science and Technology (Project No. 2023ZD0403801). The data that supports the findings of this study are available in the Supporting Information of this article. Data S1-S2 Figure S1-S9 Table S1-S3 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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