Molecular Pathogens, 2025, Vol.16, No.6, 285-293 http://microbescipublisher.com/index.php/mp 288 tools like EuPaGDT are available now (Chung et al., 2021), it's not just about inputting the target sequence and that's it. Efficiency, off-target risk, and whether there are small fragments related to repair (such as micro-homologous sequences) all need to be considered in advance. If the virus or bacteria with high genetic diversity are being dealt with, the design of gRNA becomes even more troublesome - it is not enough to be precise with just one reference sequence; it is also necessary to consider whether it can take effect across multiple strains to prevent pathogens from easily escaping (Zhu and Luo, 2025). 4.2 Comparison and evaluation of ex vivo and in vivo CRISPR editing efficiencies Many experiments are willing to start with in vitro editing at the beginning. After all, environmental control is easy, and protoplast processing, gene knockout, and sequence verification are relatively clear. This approach has been proven to successfully knock out the target site in invasive Aphanomyces invadans. However, it does not mean that in vivo manipulation is not used. On the contrary, when the CRISPR system is directly delivered into living tissues or infection systems, virulence changes in the real environment can also be observed. For example, it has been applied in studies related to Toxoplasma gondii or plant pathogens (Young et al., 2019; Olivares et al., 2021). However, the internal method often encounters an old problem: delivery is difficult, especially under the interference of the host's immune system and microenvironment, the efficiency may not be stable. 4.3 Phenotypic assessment of virulence loss and survival in edited pathogens The entire editing experiment cannot be considered complete without taking a look at the effect after gene knockout. Taking CRISPR-dot nanocomplexes as an example, after targeting the papG gene of pathogenic Escherichia coli in the urinary tract, it was found that the adhesion ability of the bacteria decreased significantly, the biofilm was not well formed, and the toxic power level of the animal model also weakened significantly (Gupta et al., 2021). Similarly, invasive Aeromonas bacteria lacking serine protease genes directly lose the ability to cause symptoms in fish. Therefore, by using model organisms for live infection and detecting survival rates and virulence indicators, it is not only possible to verify whether the genes have truly been knocked out, but also to visually demonstrate whether the pathogen's attacking power has been weakened. 5 Disease Resistance Restoration in the Cotton–Pathogen Interaction System 5.1 Molecular evidence of reduced pathogenicity after CRISPR interference The pathogenicity of pathogens is not constant. Some studies attempted to target some key genes of cotton leaf curling virus (CLCuVs) with CRISPR/Cas9 in cotton and found that the expression of the virus was indeed weakened. Especially when multiple guide Rnas simultaneously attack multiple regions of the virus, viral replication is hindered and the titer also drops significantly. Structural changes such as insertions and deletions, and double-strand breaks have all been detected at the molecular level (Binyameen et al., 2021). In true cotton plants, when CRISPR constructs and viruses were introduced simultaneously, the accumulation of viruses was significantly lower, indicating that it did play a certain inhibitory role (Mubarik et al., 2021). 5.2 Cotton immune responses and resistance gene expression activation Not all resistance reactions come from the cotton itself. Once the CRISPR system eliminates the virulence genes of pathogens, the defense mechanism on the cotton side will be activated more "confidently". Signaling pathways related to jasmonic acid, salicylic acid, etc., including those that can produce reactive oxygen species and activate NLR receptors, have become active (Umer et al., 2023; Zhu et al., 2023). In addition, in some experiments, CRISPR was also used to mutate regulatory elements, such as the MIR482 family. This not only strengthened the resistance of the plants, but also did not significantly drag down growth, and the disease index decreased accordingly (Zhu et al., 2022). 5.3 Field performance of cotton in response to reduced virulence in pathogens Ultimately, whether laboratory data can be reflected in field performance is the key. In greenhouse and field experiments, cotton plants carrying multiple CRISPR constructs and capable of targeting pathogenic genes such as CLCuV did indeed exhibit stronger resistance. Although not all strains showed significant improvement, the resistance rate of some transgenic materials reached 60%-70% (Binyameen et al., 2021). These changes are not
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