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Efficient and markerless gene integration with SlugCas9-HF in Kluyveromyces marxianus
- Source :
- Communications Biology, Vol 7, Iss 1, Pp 1-10 (2024)
- Publication Year :
- 2024
- Publisher :
- Nature Portfolio, 2024.
-
Abstract
- 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.
- Subjects :
- Biology (General)
QH301-705.5
Subjects
Details
- Language :
- English
- ISSN :
- 23993642
- Volume :
- 7
- Issue :
- 1
- Database :
- Directory of Open Access Journals
- Journal :
- Communications Biology
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.3ab9b841463c46389d6a0d337204a437
- Document Type :
- article
- Full Text :
- https://doi.org/10.1038/s42003-024-06487-w