Synthetic Genetic Circuits Enabled in Komagataella phaffii Through T7 RNAP/CRISPRa System

ABSTRACT The CRISPR activation (CRISPRa) transcriptional system has become a powerful synthetic biology tool for the regulation of endogenous gene expression, allowing for precise fine‐tuning of target genes through the simple modification of sgRNA sequences. In this study, we demonstrate that sgRNAs can be effectively expressed using the T7 transcription system. The insertion of tRNA sequences between PT7 and sgRNAs significantly enhances the efficiency of transcriptional activation. Furthermore, the design of PT7‐tRNA‐sgRNA arrays facilitates the multiplexed activation of genes. sgRNA expression was regulated by the Tet‐on induction system, split‐T7 system, and RNA cleavage processing by HH‐HDV, resulting in the creation of a Boolean logic gene circuit capable of performing both AND and OR logic operations. Finally, we developed a UPR self‐responsive system by utilizing endogenous promoters that are responsive to UPR signals to control the expression of T7 RNAP. This system dynamically regulates the expression of the endogenous HAC1 transcription factor, thus enhancing the secretion of heterologous proteins. The findings from this study highlight the potential of utilizing the T7 transcription system for the construction of genetic circuits, providing a practical toolkit for gene regulation in the industrial Komagataella phaffii strain.