ArticleCommunications biology2023
A DNA assembly toolkit to unlock the CRISPR/Cas9 potential for metabolic engineering.
Article in Communications biology, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed.
- Impact of carbon and nitrogen source variation on B-vitamin accumulation inBiochemistry and biophysics reports · 2026Article
- Expanding the Genetic Toolkit of Pichia kudriavzevii E1 for Enhanced Mycoprotein Production.Microbial biotechnology · 2026Article
- Mitochondrial and plasma‑membrane transport‑based engineering enables industrial‑level isocitric acid production in Yarrowia lipolytica.Microbial cell factories · 2026Article
- 10 years of CRISPR/CAS genomic engineering in Yarrowia lipolytica.Bioprocess and biosystems engineering · 2026Review
- Intron location and sequence modulate gene expression in Yarrowia lipolytica.Nucleic acids research · 2026Article
- Omics-Based Expression Cassette for Heterologous Protein Production inACS synthetic biology · 2026Article
- Engineering de novo formatotrophy in the non-model yeastNature synthesis · 2026Article
- EngineeringFrontiers in microbiology · 2026Review
- Advances in Lycopene Production: From Natural Sources to Microbial Synthesis UsingMolecules (Basel, Switzerland) · 2025Article
- Enhancing selective itaconic acid synthesis in Yarrowia lipolytica through targeted metabolite transport reprogramming.Biotechnology for biofuels and bioproducts · 2025Article
- Recent advances in genetic engineering and chemical production in yeast species.FEMS yeast research · 2025Review
- Metabolic engineering of Yarrowia lipolytica for the production and secretion of the saffron ingredient crocetin.Biotechnology for biofuels and bioproducts · 2025Article
- Emerging trends in genome integration tools for precision engineering of diverse bacterial species.Synthetic biology (Oxford, England) · 2025Review
- biGMamAct: efficient CRISPR/Cas9-mediated docking of large functional DNA cargoes at theSynthetic biology (Oxford, England) · 2025Article
- Engineered cross-feeding creates inter- and intra-species synthetic yeast communities with enhanced bioproduction.Nature communications · 2024Article
- Efficient synthesis of limonene production in Yarrowia lipolytica by combinatorial engineering strategies.Biotechnology for biofuels and bioproducts · 2024Article
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7 authors.
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Abstract
CRISPR/Cas9-based technologies are revolutionising the way we engineer microbial cells. One of the key advantages of CRISPR in strain design is that it enables chromosomal integration of marker-free DNA, eliminating laborious and often inefficient marker recovery procedures. Despite the benefits, assembling CRISPR/Cas9 editing systems is still not a straightforward process, which may prevent its use and applications. In this work, we have identified some of the main limitations of current Cas9 toolkits and designed improvements with the goal of making CRISPR technologies easier to access and implement. These include 1) A system to quickly switch between marker-free and marker-based integration constructs using both a Cre-expressing and standard Escherichia coli strains, 2) the ability to redirect multigene integration cassettes into alternative genomic loci via Golden Gate-based exchange of homology arms, 3) a rapid, simple in-vivo method to assembly guide RNA sequences via recombineering between Cas9-helper plasmids and single oligonucleotides. We combine these methodologies with well-established technologies into a comprehensive toolkit for efficient metabolic engineering using CRISPR/Cas9. As a proof of concept, we developed the YaliCraft toolkit for Yarrowia lipolytica, which is composed of a basic set of 147 plasmids and 7 modules with different purposes. We used the toolkit to generate and characterize a library of 137 promoters and to build a de novo strain synthetizing 373.8 mg/L homogentisic acid.
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