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- Bergenholm, David, 1987, et al.
(author)
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Rational gRNA design based on transcription factor binding data
- 2021
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In: Synthetic Biology. - : Oxford University Press (OUP). - 2397-7000 .- 1939-7267. ; 6:1
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Journal article (peer-reviewed)abstract
- The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has become a standard tool in many genome engineering endeavors. The endonuclease-deficient version of Cas9 (dCas9) is also a powerful programmable tool for gene regulation. In this study, we made use of Saccharomyces cerevisiae transcription factor (TF) binding data to obtain a better understanding of the interplay between TF binding and binding of dCas9 fused to an activator domain, VPR. More specifically, we targeted dCas9-VPR toward binding sites of Gcr1-Gcr2 and Tye7 present in several promoters of genes encoding enzymes engaged in the central carbon metabolism. From our data, we observed an upregulation of gene expression when dCas9-VPR was targeted next to a TF binding motif, whereas a downregulation or no change was observed when dCas9 was bound on a TF motif. This suggests a steric competition between dCas9 and the specific TF. Integrating TF binding data, therefore, proved to be useful for designing guide RNAs for CRISPR interference or CRISPR activation applications.
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| 2. |
- David, Florian, 1981, et al.
(author)
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A Perspective on Synthetic Biology in Drug Discovery and Development - Current Impact and Future Opportunities
- 2021
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In: SLAS Discovery. - : Elsevier BV. - 2472-5552 .- 2472-5560. ; 26:5, s. 581-603
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Research review (peer-reviewed)abstract
- The global impact of synthetic biology has been accelerating, because of the plummeting cost of DNA synthesis, advances in genetic engineering, growing understanding of genome organization, and explosion in data science. However, much of the discipline’s application in the pharmaceutical industry remains enigmatic. In this review, we highlight recent examples of the impact of synthetic biology on target validation, assay development, hit finding, lead optimization, and chemical synthesis, through to the development of cellular therapeutics. We also highlight the availability of tools and technologies driving the discipline. Synthetic biology is certainly impacting all stages of drug discovery and development, and the recognition of the discipline’s contribution can further enhance the opportunities for the drug discovery and development value chain.
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| 3. |
- Otto, Maximilian, 1991, et al.
(author)
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Expansion of the Yeast Modular Cloning Toolkit for CRISPR-Based Applications, Genomic Integrations and Combinatorial Libraries
- 2021
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In: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 10:12, s. 3461-3474
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Journal article (peer-reviewed)abstract
- Standardisation of genetic parts has become a topic of increasing interest over the last decades. The promise of simplifying molecular cloning procedures, while at the same time making them more predictable and reproducible has led to the design of several biological standards, one of which is modular cloning (MoClo). The Yeast MoClo toolkit provides a large library of characterised genetic parts combined with a comprehensive and flexible assembly strategy. Here we aimed to (1) simplify the adoption of the standard by providing a simple design tool for including new parts in the MoClo library, (2) characterise the toolkit further by demonstrating the impact of a BglII site in promoter parts on protein expression, and (3) expand the toolkit to enable efficient construction of gRNA arrays, marker-less integration cassettes and combinatorial libraries. These additions make the toolkit more applicable for common engineering tasks and will further promote its adoption in the yeast biological engineering community.
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