ArticleBiotechnology journal2016
EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9.
Article in Biotechnology journal, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 141 papers.
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141 citing papers in PubMed, 290 citations in OpenAlex.
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- Engineering N-acyl-homoserine lactone-based quorum-sensing circuit for dynamic regulatory control in Saccharomyces cerevisiae.Communications biology · 2025Article
- A yeast surface display platform for characterizing CAR T cell responses to cancer antigens.Nature communications · 2025Article
- A yeast mating platform for multiplex screening of fungal GPCR-ligand interactions.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
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- Complex Kinetic Models Predict β-Carotene Production and Reveal Flux Limitations in RecombinantACS synthetic biology · 2025Article
81 more citing papers are in PubMed but not listed here.
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Authors and funding
7 authors at 2 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker-free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone-MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90-100% and 60-70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113-7D) and a diploid industrial strain (Ethanol Red) for production of 3-hydroxypropionic acid, where we tested three different acetyl-CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone-MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.