Expanding the Repertoire of Selectable Markers for Aspergillus Transformation

Dave, Khyati; Prabha, V. Lakshmi; Ahuja, Manmeet; Dave, Kashyap; Tejaswini, S.; Punekar, N. S.

doi:10.1007/978-3-319-10503-1_11

Cited by 5 publications

(2 citation statements)

References 72 publications

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“…For diploid microorganisms (such as certain microbial fungi 48 ) or polyploid microorganisms (including bacterial and archaeal species 49,50 ), genetic interaction analysis is hindered by the need to repeatedly delete or mutate multiple copies of a genetic locus. Lack of functional selectable markers for genetic analysis in many filamentous fungi 51,52 and microbial parasites 53,54 also hinders the generation of multi-locus mutant strains. CRISPR-based technologies can overcome many of these limitations since Cas9 editing targets all homologous genomic loci simultaneously and allows for simultaneous multi-gene targeting (via the use of multiple sgRNAs).…”

Section: Crispr-enabled Microbial Genetic Interaction Analysismentioning

confidence: 99%

CRISPR-based genomic tools for the manipulation of genetically intractable microorganisms

2018

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Genetic manipulation of microorganisms has been crucial in understanding their biology, yet for many microbial species, robust tools for comprehensive genetic analysis were lacking until the advent of CRISPR-Cas-based gene editing techniques. In this Progress article, we discuss advances in CRISPR-based techniques for the genetic analysis of genetically intractable microorganisms, with an emphasis on mycobacteria, fungi and parasites. We discuss how CRISPR-based analyses in these organisms have enabled the discovery of novel gene functions, the investigation of genetic interaction networks and the identification of virulence factors.

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Section: Crispr-enabled Microbial Genetic Interaction Analysismentioning

confidence: 99%

CRISPR-based genomic tools for the manipulation of genetically intractable microorganisms

2018

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“…These markers usually confer drug resistance, with common drugs used for selection including hygromycin, nourseothricin, neomycin and phleomycin to name a few. Because these markers are heterologous, transformation outcomes (episomal maintenance or gene disruption) are easier to control (McDade and Cox 2001;Alderton et al 2006;Hu and Kronstad 2006;Dave et al 2015). However, using drug resistance as a marker could interfere with the study of genes involved in resistance to antifungals when both the antifungal and the compound that the resistance marker allows growth on are included in the same growth medium.…”

Section: Inroductionmentioning

confidence: 99%

Development of aCandida glabratadominant nutritional transformation marker utilizing theAspergillus nidulansacetamidase gene (amdS)

Blaylock

Wickes

et al. 2016

FEMS Yeast Research

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The gene encoding Aspergillus nidulans acetamidase (amdS) was placed under control of Candida albicans ACT1 promoter and terminator sequences and then cloned into a plasmid containing C. glabrata ARS10,CEN8 or ARS10+CEN8 sequences. All plasmids transformed C. glabrata wild-type cells to acetamide+, with the ARS-only containing plasmid transforming cells at the highest frequencies (>1.0 × 10(4) transformants μg(-1)). Plasmids were rapidly lost under non-selective conditions with the frequency dependent on chromosomal element, thus recycling the acetamide- phenotype. The amdS plasmid was used to transform a set of clinical isolates resistant to a variety of antifungal drugs. All strains were successfully transformed to the acetamide+ phenotype at high frequency, confirming that this plasmid construct could be used as a simple dominant marker on virtually any strain. Gap repair experiments demonstrated that just as in Saccharomyces cerevisiae, gap repair functions efficiently inC. glabrata, suggesting that C. glabrata has numerous similarities toS. cerevisiae with regard to ease of molecular manipulation. The amdS system is inexpensive and efficient, and combined with existing C. glabrata plasmid elements, confers a high transformation frequency for C. glabrata with a phenotype that can be easily recycled.

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