A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus:
            <i>In Vitro</i>
            -Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates

Abdallah, Qusai Al; Ge, Wenbo; Fortwendel, Jarrod R.

doi:10.1128/msphere.00446-17

Cited by 156 publications

(233 citation statements)

References 59 publications

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“…Introduction of Cas9-sgRNA RNPs with or without a donor template yielded hundreds to thousands of edited B. cinerea transformants. So far, similar approaches have been rather rarely used for CRISPR/Cas genome editing in fungi [22–24]. An advantage of the use of RNP over endogenous Cas9 and sgRNA expression is the reduced probability of potential off-target mutagenic activities of Cas9, because of its limited stability in cells [52].…”

Section: Discussionmentioning

confidence: 99%

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CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing inBotrytis cinerea

Hahn

Leisen

Bietz

et al. 2020

Preprint

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20CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse 21 organisms, enabling targeted genetic changes to be performed with unprecedented 22 37 research in B. cinerea and other fungi. 38 39 40Botrytis cinerea is a plant pathogenic ascomycete which infects more than a thousand species, 41 triggering gray mold disease which is responsible for over a billion dollars of losses in fruits, vegetables 42 and flowers every year [1]. Due to its worldwide occurrence, great economic importance and non-43 specific necrotrophic lifestyle, it has been ranked as the second most important plant pathogenic 44 fungus [2]. Control of gray mold often requires repeated treatments with fungicides, in particular 45 under high humidity conditions, but rapid adaption and resistance development of B. cinerea has 46 dramatically reduced their efficiency worldwide in many cultures, for example in strawberry fields [3]. 47After germination of a conidium on the plant surface, the fungus penetrates and invades the host, 48 rapidly killing plant cells by releasing a complex mixture of cell wall degrading enzymes, phytotoxic 49 metabolites and proteins, and by tissue acidification [4, 5]. How host cell death is induced is not fully 50 understood, but the invading hyphae seem to trigger the hypersensitive response, a plant-specific type 51 of apoptosis linked to strong defence reactions [6, 7]. Furthermore, B. cinerea releases small RNAs 52 (sRNAs) that can suppress the expression of defence-related genes in its host plants [8]. As a 53 countermeasure, plants also release sRNAs aimed to suppress fungal virulence [9]. To facilitate access 54 to genes or non-coding RNA loci that are important for pathogenesis, a gapless genome sequence of 55 B. cinerea has been published recently [10]. Considerable efforts have been made to generate tools 56 for the genetic manipulation of B. cinerea. Agrobacterium-mediated and protoplast-based 57 transformation have been developed [11][12][13], and several vectors are available which facilitate the 58 generation of mutants and strains expressing fluorescently tagged proteins for cytological studies [14]. 59Nevertheless, the generation of mutants remains time-consuming, partly because of the multinuclear 60 nature of B. cinerea, which requires several rounds of sub-cultivation to achieve homokaryosis. 61 Furthermore, the generation of multiple knock-out mutants is hampered by the lack of marker 62 recycling systems for serial gene replacements, as described in some filamentous fungi [15]. 63The application of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 64 RNA-guided Cas9 endonuclease activity has revolutionized genome editing and greatly facilitated the 65 4 genetic manipulation in a wide range of species [16]. CRISPR/Cas is based on the introduction of double 66 stranded breaks by the Cas9 endonuclease in the genome of an organism. Cas9 targeting occurs by 67 complementary sequences of a single guide RNA (sgRNA), which directs the endonu...

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Section: Discussionmentioning

confidence: 99%

“…Delivery of sgRNA can be achieved either via plasmids or by in vitro synthesized sgRNA. More recently, transformation with Cas9-sgRNA ribonucleoprotein (RNP) complexes has been successfully applied in selected fungi [22–24].…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing inBotrytis cinerea

Hahn

Leisen

Bietz

et al. 2020

Preprint

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“…As one means of minimizing off-target effects, directly transfected Cas9 protein reduces the off-target cleavage rate when compared with Cas9 expression by a plasmid or mRNA transfection in mammalian cells (16). One recent study has demonstrated that direct delivery of Cas9–gRNA ribonucleoprotein can facilitate genome editing in A. fumigatus (10). Based on these improvements, we produced a simple, efficient, and accurate site-directed mutagenesis system to investigate structure–phenotype relationships of the azole target Cyp51A.…”

Section: Discussionmentioning

confidence: 99%

“…DNA repair via homology-directed repair requires a homologous DNA template with sequence similarity to the adjacent region of the DSB locus, whereas NHEJ ligates the DSB, leading to indels in a template-independent manner. The Cas9/CRISPR system has also been successfully applied to A. fumigatus (10–12).…”

Section: Introductionmentioning

confidence: 99%

Cas9/CRISPR genome editing to demonstrate the contribution of Cyp51A Gly138Ser to azole resistance in Aspergillus fumigatus

Umeyama

Hayashi

Shimosaka

et al. 2018

Preprint

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23Azole resistance in Aspergillus fumigatus is predominantly associated with increased expression 24 of Cyp51A (lanosterol 14α-demethylase), the target enzyme of azole antifungal agents, or with 25 single-nucleotide polymorphisms (SNPs) in cyp51A. Although several SNPs that may be linked 26 to low susceptibility in azole-resistant isolates have previously been reported, few studies have 27 been conducted to conclusively demonstrate the contribution of SNPs to decreased azole 28susceptibility. An A. fumigatus strain was isolated from the sputum of a 74-year-old male 29 receiving long-term voriconazole treatment for chronic progressive pulmonary aspergillosis. 30Etest antifungal susceptibility testing showed low susceptibility to voriconazole, itraconazole, 31 and posaconazole. Nucleotide sequencing of cyp51A from this isolate revealed the mutations 32 Gly138Ser (GGC→AGC) and Asn248Lys (AAT AAA) compared with the cyp51A of 33 azole-susceptible isolates. PCR-amplified DNA fragments containing cyp51A with or without the 34 mutations of interest and a hygromycin marker were simultaneously introduced along with the 35 Cas9 protein and in vitro-synthesized single-guide RNA into protoplasts of the 36 azole-resistant/susceptible strains. Etest azole susceptibility testing of recombinant strains 37 showed an increased susceptibility via the replacement of Ser138 by glycine. In contrast, azole 38 susceptibility was slightly decreased when a Ser138 mutation was introduced into the 39 azole-susceptible strain AfS35, indicating that the serine at position 138 of Cyp51A contributes 40 to low susceptibility in the azole-resistant isolate. Genetic recombination, which has been 41 hampered thus far in clinical isolates, can now be achieved using Cas9/CRISPR genome editing. 42This technique could be useful to investigate the contribution of other SNPs of cyp51A to azole 43 resistance. 44 45

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“…Complementation of each RasGEF deletion was achieved by targeted, ectopic integration of each gene under the control of the endogenous promoter using a previously described CRISPR/Cas9 technique (Al Abdallah, Ge, & Fortwendel, ). Briefly, to generate the repair templates for complementation, the coding region plus 1 kb of native promoter for each RasGEF was PCR amplified from genomic DNA and fused to a phleomycin resistance cassette, previously amplified from the plasmid pAGRP (Fortwendel et al, ), using an overlap‐extension PCR technique (Szewczyk et al, ).…”

Section: Methodsmentioning

confidence: 99%

SH3‐class Ras guanine nucleotide exchange factors are essential forAspergillus fumigatusinvasive growth

Martín‐Vicente

Souza

Abdallah

et al. 2019

Cellular Microbiology

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Proper hyphal morphogenesis is essential for the establishment and progression of invasive disease caused by filamentous fungi. In the human pathogen Aspergillus fumigatus, signalling cascades driven by Ras and Ras‐like proteins orchestrate a wide variety of cellular processes required for hyphal growth. For activation, these proteins require interactions with Ras‐subfamily‐specific guanine nucleotide exchange factors (RasGEFs). Although Ras‐protein networks are essential for virulence in all pathogenic fungi, the importance of RasGEF proteins is largely unexplored. A. fumigatus encodes four putative RasGEFs that represent three separate classes of RasGEF proteins (SH3‐, Ras guanyl nucleotide‐releasing protein [RasGRP]–, and LTE‐class), each with fungus‐specific attributes. Here, we show that the SH3‐class and RasGRP‐class RasGEFs are required for properly timed polarity establishment during early growth and branch emergence as well as for cell wall stability. Further, we show that SH3‐class RasGEF activity is essential for polarity establishment and maintenance, a phenotype that is, at least, partially independent of the major A. fumigatus Ras proteins, RasA and RasB. Finally, loss of both SH3‐class RasGEFs resulted in avirulence in multiple models of invasive aspergillosis. Together, our findings suggest that RasGEF activity is essential for the integration of multiple signalling networks to drive invasive growth in A. fumigatus.

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A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro -Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates

Cited by 156 publications

References 59 publications

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing inBotrytis cinerea

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing inBotrytis cinerea

Cas9/CRISPR genome editing to demonstrate the contribution of Cyp51A Gly138Ser to azole resistance in Aspergillus fumigatus

SH3‐class Ras guanine nucleotide exchange factors are essential forAspergillus fumigatusinvasive growth

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