Establishment of CRISPR-STAR System to Realise Simultaneous Transcriptional Activation and Repression in Yarrowia lipolytica

The ability to regulate gene expression in multiple directions is crucial to maximise the production of microbial cell factories. However, the lack of a regulatory tool that can simultaneously activate and repress multiple genes restricts the manipulation diversity of Yarrowia lipolytica, which is an industrial workhorse for bioproduction. To address this issue, we developed a CRISPR scaffold RNAs (scRNAs)-mediated transcriptional activation and repression (CRISPR-STAR) platform. Firstly, we evaluated different methods for bidirectional regulation using CRISPR on both endogenous and synthetic promoters in Y. lipolytica, and chose the utilisation of orthogonal scRNAs to recruit activation and inhibition domains. Secondly, CRISPR-STAR was optimised by the introduction of alternative dCas proteins, scRNA structures and activators. 2.6-fold and 54.9-fold activation were achieved for synthetic and endogenous promoters, respectively, when the VPR transcriptional activator was recruited via MS2 hairpin. The repression of several genes was successfully achieved, with repression levels ranging from 3% to 32%, when the MXI1 transcriptional repressor was recruited via PP7 hairpin. Finally, CRISPR-STAR was applied to enhance fatty alcohol production by activating the FAR gene (encodes fatty acyl-CoA reductase) and repression of the PEX10 gene (encodes an integral membrane protein required for peroxisome biogenesis and matrix protein import). Compared to the non-targeting control, the bidirectionally regulated strain showed a 55.7% increase in yield to 778.8 mg/L. Our findings demonstrate that the CRISPR-STAR platform enables multi-mode regulation of genes, offering engineering opportunities to improve the productive performance of Y. lipolytica.