Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a
| Autor: | René Verwaal, Klaudia W Ciurkot, Thomas E. Gorochowski, Johannes Andries Roubos |
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| Rok vydání: | 2021 |
| Předmět: |
AcademicSubjects/SCI00010
CRISPR-Associated Proteins Green Fluorescent Proteins Nuclear Localization Signals Saccharomyces cerevisiae Down-Regulation BrisSynBio Computational biology Biology 03 medical and health sciences 0302 clinical medicine Protein Domains Genetics NLS CRISPR Guide RNA Promoter Regions Genetic Gene 030304 developmental biology Trans-activating crRNA Regulation of gene expression 0303 health sciences Reporter gene Endodeoxyribonucleases Bristol BioDesign Institute beta Carotene biology.organism_classification Gene Expression Regulation RNA RNA Polymerase II CRISPR-Cas Systems Synthetic Biology and Bioengineering 030217 neurology & neurosurgery |
| Zdroj: | Ciurkot, K W, Gorochowski, T E, Roubos, J A & Verwaal, R 2021, ' Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a ', Nucleic Acids Research, vol. 49, no. 13, gkab529, pp. 7775-7790 . https://doi.org/10.1093/nar/gkab529 Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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