A standardized toolkit for genetic engineering of CTG clade yeasts

Defosse, Tatiana A.; Courdavault, Vincent; Coste, Alix T.; Clastre, Marc; Bernonville, Thomas Dugé de; Godon, Charlotte; Vandeputte, Patrick; Lanoue, Arnaud; Touzé, Antoine; Linder, Tomas; Droby, Samir; Rosa, Carlos A.; Sanglard, Dominique; d’Enfert, Christophe; Bouchara, Jean‐Philippe; Giglioli-Guivarc’h, Nathalie; Papon, Nicolas

doi:10.1016/j.mimet.2017.11.015

Cited by 18 publications

(15 citation statements)

References 14 publications

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“…tropicalis. We therefore adapted constructs designed by Defosse et al (42), who previously identified suitable promoters and terminators for use in C. tropicalis.…”

Section: Resultsmentioning

confidence: 99%

Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple Candida Species

Lombardi

Oliveira-Pacheco

Butler

2019

mSphere

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Many Candida species that cause infection have diploid genomes and do not undergo classical meiosis. The application of clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) gene editing systems has therefore greatly facilitated the generation of gene disruptions and the introduction of specific polymorphisms. However, CRISPR methods are not yet available for all Candida species. We describe here an adaption of a previously developed CRISPR system in Candida parapsilosis that uses an autonomously replicating plasmid. Guide RNAs can be introduced in a single cloning step and are released by cleavage between a tRNA and a ribozyme. The plasmid also contains CAS9 and a selectable nourseothricin SAT1 marker. It can be used for markerless editing in C. parapsilosis, C. orthopsilosis, and C. metapsilosis. We also show that CRISPR can easily be used to introduce molecular barcodes and to reintroduce wild-type sequences into edited strains. Heterozygous mutations can be generated, either by careful selection of the distance between the polymorphism and the Cas9 cut site or by providing two different repair templates at the same time. In addition, we have constructed a different autonomously replicating plasmid for CRISPR-Cas9 editing in Candida tropicalis. We show that editing can easily be carried out in multiple C. tropicalis isolates. Nonhomologous end joining (NHEJ) repair occurs at a high level in C. metapsilosis and C. tropicalis. IMPORTANCE Candida species are a major cause of infection worldwide. The species associated with infection vary with geographical location and with patient population. Infection with Candida tropicalis is particularly common in South America and Asia, and Candida parapsilosis infections are more common in the very young. Molecular methods for manipulating the genomes of these species are still lacking. We describe a simple and efficient CRISPR-based gene editing system that can be applied in the C. parapsilosis species group, including the sister species Candida orthopsilosis and Candida metapsilosis. We have also constructed a separate system for gene editing in C. tropicalis.

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“…tropicalis. We therefore adapted constructs designed by Defosse et al (42), who previously identified suitable promoters and terminators for use in C. tropicalis.…”

Section: Resultsmentioning

confidence: 99%

Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple Candida Species

Lombardi

Oliveira-Pacheco

Butler

2019

mSphere

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“…We found that the pCP-tRNA plasmid failed to generate nourseothricin resistant transformants of C. tropicalis . We therefore adapted constructs designed by Defosse et al (40) who identified suitable promoters and terminators for use in C. tropicalis . We first replaced the GFP gene in pAYCU268 from Defosse et al (40) with CAS9 from Vyas et al (25), placing CAS9 under the control of the TEF1 promoter from Meyerozyma guilliermondii .…”

Section: Resultsmentioning

confidence: 99%

“…We therefore adapted constructs designed by Defosse et al (40) who identified suitable promoters and terminators for use in C. tropicalis . We first replaced the GFP gene in pAYCU268 from Defosse et al (40) with CAS9 from Vyas et al (25), placing CAS9 under the control of the TEF1 promoter from Meyerozyma guilliermondii . pAYCU268 expresses SAT1 from the C. dubliniensis TEF1 promoter, and is designed to integrate randomly in the C. tropicalis genome.…”

Section: Resultsmentioning

confidence: 99%

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Plasmid-based CRISPR-Cas9 gene editing in multipleCandidaspecies

Lombardi

Oliveira-Pacheco

Butler

2019

Preprint

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“…However, genetic manipulation has often been time-consuming and cumbersome in fungi, particularly in species with diploid genomes that lack a sexual cycle. A low efficiency of transformation and/or homologous recombination, the absence of natural plasmids, and the lack of cloning vectors or the limited number of dominant markers available for selection are additional factors hampering this process [5,6].…”

Section: Introductionmentioning

confidence: 99%

The CRISPR toolbox in medical mycology: State of the art and perspectives

2020

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Fungal pathogens represent a major human threat affecting more than a billion people worldwide. Invasive infections are on the rise, which is of considerable concern because they are accompanied by an escalation of antifungal resistance. Deciphering the mechanisms underlying virulence traits and drug resistance strongly relies on genetic manipulation techniques such as generating mutant strains carrying specific mutations, or gene deletions. However, these processes have often been time-consuming and cumbersome in fungi due to a number of complications, depending on the species (e.g., diploid genomes, lack of a sexual cycle, low efficiency of transformation and/or homologous recombination, lack of cloning vectors, nonconventional codon usage, and paucity of dominant selectable markers). These issues are increasingly being addressed by applying clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 mediated genetic manipulation to medically relevant fungi. Here, we summarize the state of the art of CRISPR-Cas9 applications in four major human fungal pathogen lineages: Candida spp., Cryptococcus neoformans, Aspergillus fumigatus, and Mucorales. We highlight the different ways in which CRISPR has been customized to address the critical issues in different species, including different strategies to deliver the CRISPR-Cas9 elements, their transient or permanent expression, use of codon-optimized CAS9, and methods of marker recycling and scarless editing. Some approaches facilitate a more efficient use of homology-directed repair in fungi in which nonhomologous end joining is more commonly used to repair double-strand breaks (DSBs). Moreover, we highlight the most promising future perspectives, including gene drives, programmable base editors, and nonediting applications, some of which are currently available only in model fungi but may be adapted for future applications in pathogenic species. Finally, this review discusses how the further evolution of CRISPR technology will allow mycologists to tackle the multifaceted issue of fungal pathogenesis.

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A standardized toolkit for genetic engineering of CTG clade yeasts

Cited by 18 publications

References 14 publications

Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple Candida Species

Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple Candida Species

Plasmid-based CRISPR-Cas9 gene editing in multipleCandidaspecies

The CRISPR toolbox in medical mycology: State of the art and perspectives

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