Scarless gene deletion in methylotrophic Hansenula polymorpha by using mazF as counter-selectable marker

2015 ◽  
Vol 468 ◽  
pp. 66-74 ◽  
Author(s):  
Panpan Song ◽  
Sha Liu ◽  
Xuena Guo ◽  
Xuejing Bai ◽  
Xiuping He ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Selectable Marker ◽  
Hansenula Polymorpha

scholarly journals Restructured Lactococcus lactis strains with emergent properties constructed by a novel highly efficient screening system

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Fulu Liu ◽  
Yating Zhang ◽  
Wanjin Qiao ◽  
Duolong Zhu ◽  
Haijin Xu ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Large Scale ◽  
Gene Deletion ◽  
Selectable Marker ◽  
Genome Reduction ◽  
Emergent Properties ◽  
Metabolic Capacity ◽  
Efficient System ◽  
Cell Factories ◽  
Efficient Screening

Abstract Background After 2.83% genome reduction in Lactococcus lactis NZ9000, a good candidate host for proteins production was obtained in our previous work. However, the gene deletion process was time consuming and laborious. Here, we proposed a convenient gene deletion method suitable for large-scale genome reduction in L. lactis NZ9000. Results Plasmid pNZ5417 containing a visually selectable marker PnisZ-lacZ was constructed, which allowed more efficient and convenient screening of gene deletion mutants. Using this plasmid, two large nonessential DNA regions, L-4A and L-5A, accounting for 1.25% of the chromosome were deleted stepwise in L. lactis 9k-3. When compared with the parent strain, the mutant L. lactis 9k-5A showed better growth characteristics, transformability, carbon metabolic capacity, and amino acids biosynthesis. Conclusions Thus, this study provides a convenient and efficient system for large-scale genome deletion in L. lactis through application of visually selectable marker, which could be helpful for rapid genome streamlining and generation of restructured L. lactis strains that can be used as cell factories.

scholarly journals High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins

2019 ◽  
Author(s):  
Peter Jan Vonk ◽  
Natalia Escobar ◽  
Han A. B. Wösten ◽  
Luis G. Lugones ◽  
Robin A. Ohm
Keyword(s):  
Homologous Recombination ◽  
High Throughput ◽  
Gene Deletion ◽  
Selectable Marker ◽  
Schizophyllum Commune ◽  
Model Systems ◽  
Targeted Gene Deletion ◽  
Repair Template ◽  
Low Incidence ◽  
Non Homologous End Joining

AbstractEfficient gene deletion methods are essential for the high-throughput study of gene function. Compared to most ascomycete model systems, gene deletion is more laborious in mushroom-forming basidiomycetes due to the relatively low incidence of homologous recombination (HR) and relatively high incidence of non-homologous end-joining (NHEJ). Here, we describe the use of pre-assembled Cas9-sgRNA ribonucleoproteins (RNPs) to efficiently delete the homeodomain transcription factor gene hom2 in the mushroom-forming basidiomycete Schizophyllum commune by replacing it with a selectable marker. All components (Cas9 protein, sgRNA, and repair template with selectable marker) were supplied to wild type protoplasts by PEG-mediated transformation, abolishing the need to optimize the expression of cas9 and sgRNAs. A Δku80 background further increased the efficiency of gene deletion. A repair template with homology arms of 250 bp was sufficient to induce homologous recombination, whereas 100 bp was not. This is the first report of the use of pre-assembled Cas9 RNPs in a mushroom-forming basidiomycete and this approach may also improve the genetic accessibility of non-model species.

scholarly journals Strains and Strategies for Large-Scale Gene Deletion Studies of the Diploid Human Fungal Pathogen Candida albicans

2005 ◽  
Vol 4 (2) ◽  
pp. 298-309 ◽  
Author(s):  
Suzanne M. Noble ◽  
Alexander D. Johnson
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Large Scale ◽  
Gene Deletion ◽  
Selectable Marker ◽  
Wild Type ◽  
Position Effects ◽  
Human Fungal Pathogen ◽  
Chromosome Position ◽  
Knockout Mutants

ABSTRACT Candida albicans is the most common human fungal pathogen and causes significant morbidity and mortality worldwide. Nevertheless, the basic principles of C. albicans pathogenesis remain poorly understood. Of central importance to the study of this organism is the ability to generate homozygous knockout mutants and to analyze them in a mammalian model of pathogenesis. C. albicans is diploid, and current strategies for gene deletion typically involve repeated use of the URA3 selectable marker. These procedures are often time-consuming and inefficient. Moreover, URA3 expression levels—which are susceptible to chromosome position effects—can themselves affect virulence, thereby complicating analysis of strains constructed with URA3 as a selectable marker. Here, we describe a set of newly developed reference strains (leu2Δ/leu2Δ, his1Δ/his1Δ; arg4Δ/arg4Δ, his1Δ/his1Δ; and arg4Δ/arg4Δ, leu2Δ/leu2Δ, his1Δ/his1Δ) that exhibit wild-type or nearly wild-type virulence in a mouse model. We also describe new disruption marker cassettes and a fusion PCR protocol that permit rapid and highly efficient generation of homozygous knockout mutations in the new C. albicans strains. We demonstrate these procedures for two well-studied genes, TUP1 and EFG1, as well as a novel gene, RBD1. These tools should permit large-scale genetic analysis of this important human pathogen.

FMR2 gene deletion as a cause of non-specific mental retardation and autistic behavior in two brothers

2010 ◽  
Vol 41 (02) ◽  
Author(s):  
GM Stettner ◽  
B Auber ◽  
M Shoukier ◽  
C Höger ◽  
K Brockmann
Keyword(s):  
Mental Retardation ◽  
Gene Deletion ◽  
Autistic Behavior

Long-term follow-up of a family with a large AIP gene deletion: variable phenotypes and challenges in the management

2017 ◽  
Author(s):  
Pedro Marques ◽  
Mary Dang ◽  
Arla Ogilvie ◽  
Helen Storr ◽  
Michael Powell ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Long Term Follow Up ◽  
Aip Gene
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