ArticleCommunications biology2024
Efficient and markerless gene integration with SlugCas9-HF in Kluyveromyces marxianus.
Article in Communications biology, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed.
- Development of a CRISPR/Cas12a genome editing toolbox inSynthetic and systems biotechnology · 2026Article
- Cidea Targeting Protects Cochlear Hair Cells and Hearing Function From Drug- and Noise-Induced Damage.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2026Article
- Intergeneric chromosomal transfer in yeast results in improved phenotypes and widespread transcriptional responses.Nature communications · 2026Article
- A CRISPR/Cas9-based genome-editing platform enabling efficient and precise gene replacement in Lipomyces starkeyi.FEMS yeast research · 2026Article
- Recent advances in microbial fermentation for the production of leghemoglobin.World journal of microbiology & biotechnology · 2025Review
- A toolkit for facilitating markerless integration of expression cassettes in Komagataella phaffii via CRISPR/Cas9.Microbial cell factories · 2025Article
- Alien Chromosome Serves as a Novel Platform for Multiple Gene Expression inMicroorganisms · 2025Article
- Metabolic engineering of yeast to efficiently synthesize heme and hemoproteins: recent advance and prospects.FEMS yeast research · 2025Review
- From genetic constraints to process hurdles: A review ofCurrent research in food science · 2025Review
Corrections and comments
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Authors and funding
6 authors.
Funding
Abstract
The nonconventional yeast Kluyveromyces marxianus has potential for industrial production, but the lack of advanced synthetic biology tools for precise engineering hinders its rapid development. Here, we introduce a CRISPR-Cas9-mediated multilocus integration method for assembling multiple exogenous genes. Using SlugCas9-HF, a high-fidelity Cas9 nuclease, we enhance gene editing precision. Specific genomic loci predisposed to efficient integration and expression of heterologous genes are identified and combined with a set of paired CRISPR-Cas9 expression plasmids and donor plasmids to establish a CRISPR-based biosynthesis toolkit. This toolkit enables genome integration of large gene modules over 12 kb and achieves simultaneous quadruple-locus integration in a single step with 20% efficiency. As a proof-of-concept, we apply the toolkit to screen for gene combinations that promote heme production, revealing the importance of HEM4Km and HEM12Sc. This CRISPR-based toolkit simplifies the reconstruction of complex pathways in K. marxianus, broadening its application in synthetic biology.
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Registered trials
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