## 4 Application of CRISPR/Cas9-Based Genome Editing Technology in _A. orgyaeae_ and _A. niger_
### CRISPR/Cas9-Based Genome Editing Technology for _A. niger_  
_A. niger_ possesses exceptional tolerance to extreme environmental conditions, high production economy, robust fermentation stability, and superior food safety; it has been granted generally recognized as safe (GRAS) status by the U.S. Food and Drug Administration (FDA) [32; 147], so it is a suitable cell factory for diverse biotechnological applications [40]. In 2016, [PERSON] et al. first successfully established the CRISPR-Cas9 system for genetic engineering in _A. niger_[148]. To disrupt the catabolism of galactaric acid in _A. niger_ and thereby generate an engineered strain capable of the efficiently producing galactaric acid, they identified seven genes--putatively encoding metabolic enzymes and transport proteins--through RNA sequencing, and then they deleted these genes by using CRISPR/Cas9 technology in conjunction with in vitro synthesized sgRNA. Subsequently, the same group successively achieved the effective deletion of _gluD_ and _gluF_ genes using the identical CRISPR/Cas9 strategy, which encode the NADPH-requiring 2-keto-L-guionate reductase and NADPH-requiring 5-keto-D-gluconate reductase, respectively [149; 150]. On this basis, [PERSON] et al. further developed an optimized CRISPR/Cas9 genome editing method for _A. niger_, based on in vitro-assembled Cas9/gRNA ribonucleoprotein (RNP) complexes, and achieved a 100% targeting efficiency for a single genomic target [151]. In terms of site-specific gene insertion, [PERSON] et al. established an efficient genetic engineering toolbox for _A. niger_, which combines the GoldenMOCS cloning system (a Golden Gate-derived modular cloning strategy) with a CRISPR/Cas9-mediated integration approach to enable site-specific insertion of heterologous expression cassettes at the _pyrG_ locus [152]. This system used a transiently expressed Cas9 (via a size-reduced AMA1 plasmid that is readily lost) and a split-_pyrG_ marker for direct selection, achieving a high integration efficiency of up to 100%. Beyond targeted gene editing, transcriptional regulation of gene expression also serves as a pivotal approach for constructing and optimizing cell factories. The Tet-on system is an effective method for functional analysis of essential genes, allowing for precise modulation of gene expression levels in response to inducer concentrations [153; 154]. In recent years, researchers have focused on integrating CRISPR/Cas9 genome editing technology with the Tet-on regulatory system in _A. niger_, aiming to quantitatively elucidate the impacts of differential expression levels of target genes on product biosynthesis. For example, [PERSON] et al. employed CRISPR-based genome editing to integrate the titratable Tet-on expression system upstream of _ageB_, _secG_, and _geaB_ in _A. niger_. They utilized this strategy to investigate the functional associations between protein secretion, filamentous growth, and organic acid production [155]. Moreover, [PERSON] et al. replaced the native promoter of the _pyrG_ gene in _A. niger_ with a Tet-on inducible promoter through in situ promoter substitution mediated by CRISPR/Cas9 genome editing [156]. By titrating the concentration of the inducer doxycycline (Dox), they established a quantitative relationship between the differential expression levels of _pyrG_ and the performance of citric acid fermentation, including titers and productivity. Very recently, a CRISPR/Cas9-based visual toolkit enabling multiplex integration at specific genomic loci has been established in _A. niger_[157], which allows simultaneous insertion of recombinant genes into multiple high-expression loci through the combined application of a visual multigene editing system (VMS) and the CRISPR-HDR system.