ArticleFEMS yeast research2022
Protocols for marker-free gene knock-out and knock-down in Kluyveromyces marxianus using CRISPR/Cas9.
Article in FEMS yeast research, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed, 18 citations in OpenAlex.
- Metabolic EngineeringJournal of fungi (Basel, Switzerland) · 2026Article
- Signatures of Innovation and Selection in the Extremotolerant Yeast Kluyveromyces marxianus.Genome biology and evolution · 2026Article
- Development of a CRISPR/Cas12a genome editing toolbox inSynthetic and systems biotechnology · 2026Article
- Adaptive laboratory evolution of maltose utilization in wild Irish isolates of Saccharomyces eubayanus.FEMS yeast research · 2026Article
- A Study on the Extraction, Fermentation Condition Optimization, and Antioxidant Activity Assessment of Polysaccharides Derived fromFoods (Basel, Switzerland) · 2025Article
- Gene disruption via a transient hypercompact CRISPR-AsCas12f1 system in Kluyveromyces marxianus.Biotechnology letters · 2025Article
- Efficient and markerless gene integration with SlugCas9-HF in Kluyveromyces marxianus.Communications biology · 2024Article
- The superior growth of Kluyveromyces marxianus at very low potassium concentrations is enabled by the high-affinity potassium transporter Hak1.FEMS yeast research · 2024Article
- RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering ofMetabolic engineering communications · 2022Article
- Identification of a novel gene required for competitive growth at high temperature in the thermotolerant yeastMicrobiology (Reading, England) · 2022Article
Corrections and comments
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
There is increased interest in strain engineering in the food and industrial yeast Kluyveromyces marxianus and a number of CRISPR/Cas9 systems have been described and used by different groups. The methods that we developed allow for very rapid and efficient inactivation of target genes using the endogenous DNA repair mechanisms of the cell. The strains and plasmids that we use are freely available, and here we provide a set of integrated protocols to easily inactivate genes and to precisely integrate DNA fragments into the genome, for example for promoter replacement, allelic swaps or introduction of point mutations. The protocols use the Cas9/gRNA expression plasmid pUCC001 and Golden Gate assembly for molecular cloning of targeting sequences. A genome-wide set of target sequences is provided. Using these plasmids in wild-type strains or in strains lacking non-homologous end-joining (NHEJ) DNA repair, the first set of protocols explain how to introduce indels (NHEJ-mediated) or precise deletions (homology-dependent repair (HDR)-mediated) at precise targets. The second set of protocols describe how to swap a promoter or coding sequence to yield a reprogrammed gene. The methods do not require the use of dominant or auxotrophic marker genes and thus the strains generated are marker-free. The protocols have been tested in multiple K. marxianus strains, are straightforward and can be carried out in any molecular biology laboratory without specialized equipment.
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