Molecular Pathogens, 2025, Vol.16, No.6, 285-293 http://microbescipublisher.com/index.php/mp 287 candidate genes, but also directly observe the performance changes of pathogens after they are destroyed - whether the virulence is weakened and whether they are still responsive to drugs, all of these can be seen (Gupta et al., 2019; Wei and Li, 2023). Especially now, with the advent of multiple Sgrnas and more flexible vector systems, multiple targets can be processed at once, thus enabling faster localization of those truly critical genetic factors (Allemailem, 2024). However, it's not always possible to handle it smoothly. It still depends on the characteristics of the target strain. Figure 1 Phenotypic effects of large chromosome fragment deletion induced by CRISPR-Cas9 and Cas12a systems (Adopted from Yuan et al., 2024) 3.3 Technical challenges and optimization strategies in microbial genome editing Despite this, the application of CRISPR in microorganisms has not been as smooth as "imagined". One of the most common problems is that the homologous recombination efficiency of some fungi is too low, making it difficult to achieve precise replacement after cutting. Another is the off-target effect, which may accidentally damage other genes (Li et al., 2019; Allemailem, 2024). Another practical problem is that editing tools simply cannot enter the cells of certain pathogens. The current solutions to these bottlenecks mainly rely on detail optimization, such as designing Sgrnas with higher specificity, switching to the high-fidelity Cas9 version, or simply skipping the vector and directly delivering RNP complexes for a short time (Ma et al., 2025). Although these technological improvements are still in progress, this step is indispensable if CRISPR is to be truly popularized in various microbial pathogens (Yang et al., 2025). 4 Design and Validation of CRISPR Targets in Drug-Resistant Genes 4.1 Principles of gRNA design targeting virulence genes Designing gRNA is no easy task, especially when it comes to targeting sites directly related to the virulence of pathogens. Key fragments such as adhesion factors or effector proteins are usually the preferred targets. Although
RkJQdWJsaXNoZXIy MjQ4ODYzNA==