Genetic mutations and natural variations are the basis for analysis of crop gene function and underlie the formation of important agronomic traits (Song et al., 2022). In crop-breeding research, CRISPR/Cas genome editing approaches can overcome the disadvantages of random mutations induced by physical/ chemical mutagenesis and T-DNA insertion, enabling precisely targeted editing of specific genomic sites. To date, Cas9 (Jiang et al., 2024, He et al., 2024), Cas12a (Cpf1) (Bandyopadhyay et al., 2020, Li et al., 2023), base editors (adenine base editor [ABE] and cytosine base editor [CBE]) (Li et al., 2020, Luo et al., 2023), and prime editing systems (Butt et al., 2020, Lu et al., 2021) have been successfully established in various plant species and crops. However, there are still many obstacles to efficient gene editing in crops with complex genomes, such as cotton. First, the expression levels of Cas protein and sgRNAs are constrained by the species-specificity of their promoters, and the different promoters used to drive their expression result in significantly different editing efficiencies in cotton (Long et al., 2018, Wang et al., 2018, Zhu et al., 2018). Second, technical bottlenecks in the design and delivery of sgRNAs still limit our ability to target multiple cotton genes simultaneously and to obtain large numbers of random mutations through construction of sgRNA libraries (Ramadan et al., 2021). Finally, genotype restrictions currently limit gene editing to a small number of cotton varieties, and these still require labor-intensive tissue culture procedures.

Virus-induced genome editing (VIGE) has emerged as a potential means of addressing these challenges. Plant viral vectors, characterized by autonomous replication and mobility within host cells, can serve as ideal vehicles for transient transformation. Plant viruses are primarily categorized as RNA or DNA viruses on the basis of their genetic material. Recent studies have demonstrated that engineered plant viruses assembled into plant expression vectors can achieve gene silencing and overexpression through Agrobacterium-mediated infiltration (Jian et al., 2017, Chen et al., 2022) and can even enable genome editing (Zhang et al., 2022, Lee et al., 2024, Wu et al., 2024, Wang et al., 2024). Cotton leaf crumple virus (CLCrV) is a DNA virus that can systemically and persistently infect cotton. Replacement of its coat protein with short gene-silencing fragments can suppress endogenous gene expression and induce gene-silencing phenotypes (Gu et al., 2014). Previous studies in our research group demonstrated the feasibility of CLCrV for cotton gene editing. Replacement of the CLCrV-A coat protein with a truncated Arabidopsis or cotton endogenous U6 promoter driving sgRNA expression enabled targeted gene editing in Cas9-overexpressing (Cas9-OE) Arabidopsis and cotton plants, with observable mutant phenotypes in visual marker genes (Lei et al., 2021, Lei et al., 2022, Lei et al., 2023, Lei et al., 2024). However, our previous studies focused mainly on the construction and preliminary validation of cotton VIGE systems. By contrast, little is known about how Cas9-expressing lines driven by different promoters function as VIGE receptors to affect the editing efficiency of target genes. For example, the 35S promoter is widely used in dicots, but the intensity of its phloem-specific expression may be insufficient to sustain prolonged Cas9 activity, and ubiquitin or tissue-specific promoters might enhance editing efficiency by improving the spatiotemporal profile of Cas9 expression (Zheng et al., 2020, Wolabu et al., 2020). In addition, studies have demonstrated that pooled inoculation of viral particles carrying different sgRNAs can enable multiplex gene editing in wheat (Wang et al., 2022), but the potential of CLCrV-mediated VIGE systems for multi-target editing in cotton remains to be explored. Likewise, a CLCrV-mediated base-editing system has not yet been established in cotton, and the potential for such a system to enable targeted base editing requires further investigation. A recent report showed that the delivery of complete CBEs into wild-type Nicotiana benthamiana via potato virus X (PVX) facilitated C-to-T base conversion at the NbTOM1 target site (Ariga et al., 2020). Similarly, Liu et al. (2022) assembled sgRNAs targeting Arabidopsis AtPDS3, AtCLA1, and AtCESA3 onto tobacco rattle virus (TRV), achieving both targeted C-to-T base conversion and heritable gene editing in CBE-overexpressing Arabidopsis plants. These virus-mediated editing tools provide streamlined technical platforms for screening of high-efficiency sgRNAs and investigation of plant gene functions.

Unlike PVX, CLCrV cannot deliver a complete set of CRISPR/Cas components in cotton (Zhao et al., 2022). Therefore, in the present study, we used previously obtained transgenic cotton materials, including nCas9-TadA7.10-OE, ubiquitin-promoter-driven Cas9 (ProUbi::Cas9), and 35S-promoter-driven Cas9 (Pro35s::Cas9) lines, as VIGE receptors, then constructed a CLCrV-mediated CRISPR system and assessed its efficacy and efficiency. Through this approach, we aimed to establish a streamlined gene-editing platform that would provide robust technical support for functional genomics and molecular breeding research in cotton.