Implementation of a CRISPR-Based System for Gene Regulation in
            <i>Candida albicans</i>

Román, Elvira; I, Coman; Prieto, Daniel; Alonso-Monge, Rebeca; Pla, Jesús

doi:10.1128/msphere.00001-19

Cited by 40 publications

(44 citation statements)

References 47 publications

(72 reference statements)

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“…In our study, the % INDELs (8.9–17.1%) resulting from NHEJ after CRISPR/Cas9 gene editing on Bg AIF exon 4 locus did not correlate with its transcript reduction (~ 50%) in all experimental samples. Nonetheless, alternative mechanisms could be used as the fusion of suppressors with a ‘dead’ Cas9 which enables gene regulation and increase the level of repression of the target gene [ 66 ].…”

Section: Discussionmentioning

confidence: 99%

Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor

et al. 2020

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Background Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro. Methods Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed. Results Gene knockout manipulation induced gene-disrupting indels, frequently 1–2 bp insertions and/or 8–30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5 ± 20.2% SD, P ≤ 0.05, n = 12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66 ± 0.10, ΔBgAIF, 2.30 ± 0.22 (P ≤ 0.01). Conclusions The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

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Section: Discussionmentioning

confidence: 99%

Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor

et al. 2020

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“…The faster and cheaper genetic manipulation of fungal genome and expression facilitated by CRISPR-Cas9 is likely to have a profound impact on medical mycology. Loss of function, gene activation, and gene inhibition strategies can be scaled up as CRISPR-Cas9 libraries [95][96][97], and large-scale library screens can facilitate the identification of new targets for antifungal therapy, of which there is a desperate need [98].…”

Section: Perspectives and Future Directionsmentioning

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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“…While most of these Cas9 variants have demonstrated high on-target mutation efficiencies in S. cerevisiae, their application can readily be extended to other fungal pathogens. Recently, Roman et al employed dCas9 fused with transcriptional activator Gal4 and/or VP64 and repressor Nrg1 to regulate expression of catalase gene CAT1 in Candida albicans [33]. Wensing and colleagues also developed an improved dCas9 system fused with codon optimised repressor domain of mammalian repressor Mxi1.…”

Section: Crispr/cas Gene Editingmentioning

confidence: 99%

Advances in Molecular Tools and In Vivo Models for the Study of Human Fungal Pathogenesis

2020

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Pathogenic fungi represent an increasing infectious disease threat to humans, especially with an increasing challenge of antifungal drug resistance. Over the decades, numerous tools have been developed to expedite the study of pathogenicity, initiation of disease, drug resistance and host-pathogen interactions. In this review, we highlight advances that have been made in the use of molecular tools using CRISPR technologies, RNA interference and transposon targeted mutagenesis. We also discuss the use of animal models in modelling disease of human fungal pathogens, focusing on zebrafish, the silkworm, Galleria mellonella and the murine model.

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Implementation of a CRISPR-Based System for Gene Regulation in Candida albicans

Cited by 40 publications

References 47 publications

Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor

Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor

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

Advances in Molecular Tools and In Vivo Models for the Study of Human Fungal Pathogenesis

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