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Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a.

ABSTRACT: CRISPR Cas12a is an RNA-programmable endonuclease particularly suitable for gene regulation. This is due to its preference for T-rich PAMs that allows it to more easily target AT-rich promoter sequences, and built-in RNase activity which can process a single CRISPR RNA array encoding multiple spacers into individual guide RNAs (gRNAs), thereby simplifying multiplexed gene regulation. Here, we develop a flexible dCas12a-based CRISPRi system for Saccharomyces cerevisiae and systematically evaluate its design features. This includes the role of the NLS position, use of repression domains, and the position of the gRNA target. Our optimal system is comprised of dCas12a E925A with a single C-terminal NLS and a Mxi1 or a MIG1 repression domain, which enables up to 97% downregulation of a reporter gene. We also extend this system to allow for inducible regulation via an RNAP II-controlled promoter, demonstrate position-dependent effects in crRNA arrays, and use multiplexed regulation to stringently control a heterologous β-carotene pathway. Together these findings offer valuable insights into the design constraints of dCas12a-based CRISPRi and enable new avenues for flexible and efficient gene regulation in S. cerevisiae.

SUBMITTER: Ciurkot K

PROVIDER: S-EPMC8287914 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a.

Ciurkot Klaudia K Gorochowski Thomas E TE Roubos Johannes A JA Verwaal René R

Nucleic acids research 20210701 13

CRISPR Cas12a is an RNA-programmable endonuclease particularly suitable for gene regulation. This is due to its preference for T-rich PAMs that allows it to more easily target AT-rich promoter sequences, and built-in RNase activity which can process a single CRISPR RNA array encoding multiple spacers into individual guide RNAs (gRNAs), thereby simplifying multiplexed gene regulation. Here, we develop a flexible dCas12a-based CRISPRi system for Saccharomyces cerevisiae and systematically evaluate ...[more]

PMID: 34197613

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Project description:Saccharomyces cerevisiae is a widely used strain for ethanol fermentation; meanwhile, efficient utilization of glucose could effectively promote ethanol production. The PFK1 gene is a key gene for intracellular glucose metabolism in S. cerevisiae. Our previous work suggested that although deletion of the PFK1 gene could confer higher oxidative tolerance to S. cerevisiae cells, the PFK1Δ strain was prone to contamination by other microorganisms. High interspecies microbial competition ability is vital for the growth and survival of microorganisms in co-cultures. The result of our previous studies hinted us a reasonable logic that the EMP (i.e., the Embden-Meyerhof-Parnas pathway, the glycolytic pathway) key gene PFK1 could be involved in regulating interspecies competitiveness of S. cerevisiae through the regulation of glucose utilization and ethanol production efficiency. The results suggest that under 2% and 5% glucose, the PFK1Δ strain showed slower growth than the S288c wild-type and TDH1Δ strains in the lag and exponential growth stages, but realized higher growth in the stationary stage. However, relative high supplement of glucose (10%) eliminated this phenomenon, suggesting the importance of glucose in the regulation of PFK1 in yeast cell growth. Furthermore, during the lag growth phase, the PFK1Δ strain displayed a decelerated glucose consumption rate (P < 0.05). The expression levels of the HXT2, HXT5, and HXT6 genes decreased by approximately 0.5-fold (P < 0.05) and the expression level of the ZWF1 exhibited a onefold increase in the PFK1Δ strain compared to that in the S. cerevisiae S288c wild-type strain (P < 0.05).These findings suggested that the PFK1 inhibited the uptake and utilization of intracellular glucose by yeast cells, resulting in a higher amount of residual glucose in the medium for the PFK1Δ strain to utilize for growth during the reverse overshoot stage in the stationary phase. The results presented here also indicated the potential of ethanol as a defensive weapon against S. cerevisiae. The lower ethanol yield in the early stage of the PFK1Δ strain (P < 0.001) and the decreased expression levels of the PDC5 and PDC6 (P < 0.05), which led to slower growth, resulted in the strain being less competitive than the wild-type strain when co-cultured with Escherichia coli. The lower interspecies competitiveness of the PFK1Δ strain further promoted the growth of co-cultured E. coli, which in turn activated the ethanol production efficiency of the PFK1Δ strain to antagonize it from E. coli at the stationary stage. The results presented clarified the regulation of the PFK1 gene on the growth and interspecies microbial competition behavior of S. cerevisiae and would help us to understand the microbial interactions between S. cerevisiae and other microorganisms. KEY POINTS: • PFK1Δ strain could realize reverse growth overshoot at the stationary stage • PFK1 deletion decreased ethanol yield and interspecific competitiveness • Proportion of E. coli in co-culture affected ethanol yield capacity of yeast cells.

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Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a.

Publications

Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a.

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