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RNA Polymerase III Promoters Compatible with CRISPR Gene Regulation in Saccharomyces cerevisiae
Kendreze L Holland1,2, Ines Blancher3, Marisa McKesey3
1Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States.
ACS Synthetic Biology
|August 18, 2025
Summary
Researchers characterized 20 RNA polymerase III (Pol III) promoters for CRISPR applications in yeast. Two promoters from Kluyveromyces lactis demonstrated effective CRISPR function, enabling enhanced hydrogen peroxide resistance through simultaneous gene activation and repression.
Area of Science:
- Molecular Biology
- Synthetic Biology
- Yeast Genetics
Background:
- CRISPR-(d)Cas9 technologies are vital for studying gene regulation and function in Saccharomyces cerevisiae.
- Simultaneous modulation of multiple gene targets is crucial for synthetic biology, metabolic engineering, and genetic interaction studies.
- A limited number of well-characterized RNA polymerase III (Pol III) promoters exist for single-guide RNA (sgRNA) expression in S. cerevisiae.
Purpose of the Study:
- To characterize 20 RNA Pol III promoters from various yeast and mammalian species for CRISPR-mediated gene activation and repression in S. cerevisiae.
- To identify novel Pol III promoters that can effectively drive sgRNA expression for multiplexed CRISPR applications.
- To assess the cross-species functionality and impact of promoter architecture on CRISPR system performance.
Main Methods:
- Characterization of 20 RNA Pol III promoters from different species for their ability to mediate CRISPR activation and repression in S. cerevisiae.
- Evaluation of promoter architecture and core sequence motifs influencing cross-species Pol III promoter functionality.
- Assessment of scaffold-mediated recruitment of multiple effectors to enhance promoter function.
- Testing the efficacy of identified promoters in simultaneous CRISPR-mediated activation and repression of endogenous S. cerevisiae genes.
Main Results:
- Cross-species functionality of Pol III promoters is influenced by promoter architecture and core sequence motifs.
- Two Kluyveromyces lactis Pol III promoters were identified as effective for CRISPR applications, comparable to the S. cerevisiae pSNR52 promoter.
- Scaffold-mediated recruitment can rescue poor promoter function in certain contexts.
- Non-native Pol III promoters enabled effective simultaneous CRISPR-mediated activation and repression of endogenous genes, enhancing resistance to hydrogen peroxide.
Conclusions:
- The characterized Pol III promoters demonstrate cross-species compatibility in simple eukaryotes, expanding the toolkit for yeast genetic engineering.
- These promoters are valuable for synthetic biology and phenotype engineering applications, enabling precise control of gene expression.
- The findings facilitate multiplexed CRISPR applications for complex genetic studies and metabolic engineering in yeast.
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