Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli

Nødvig, Christina Spuur; Hoof, Jakob Blæsbjerg; Kogle, Martin Engelhard; Jarczynska, Zofia Dorota; Lehmbeck, Jan; Klitgaard, Dorte K; Mortensen, Uffe Hasbro

doi:10.1016/j.fgb.2018.01.004

Cited by 155 publications

(203 citation statements)

References 47 publications

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“…In parallel, we built and tested a dSpCas9-VPR system, with a sgRNA expression cassette driven by the RNA polymerase III promoter U3 from Aspergillus fumigatus ( AfP U3 ) 30 (Supplementary note 1). In this case, four sgRNA were tested individually, targeting a window 162–342 bp from the reporter start codon, and delivered in a single AMA1-pyrG vector together with the reporter construct P elcA -mCherry .…”

Section: Resultsmentioning

confidence: 99%

CRISPR-mediated activation of biosynthetic gene clusters for bioactive molecule discovery in filamentous fungi

Roux

Woodcraft

et al. 2020

Preprint

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7Accessing the full biosynthetic potential encoded in the genomes of fungi is limited by the low 8 expression of many biosynthetic gene clusters (BGCs) under standard culture conditions. In 9 this work, we develop a fungal CRISPR activation (CRISPRa) system for targeted upregulation 10 of biosynthetic genes, which could accelerate the emerging genomics-driven approach to 11 bioactive secondary metabolite discovery. We construct a fungal CRISPR/dLbCas12a-VPR 12 system and demonstrate activation of a fluorescent reporter in Aspergillus nidulans. Then, we 13 target the native nonribosomal peptide synthetase-like (NRPS-like) gene micA in both 14 chromosomal and episomal contexts, achieving increases in production of the compound 15 microperfuranone. Finally, multi-gene CRISPRa leads to the discovery of the mic cluster 16 product as dehydromicroperfuranone. Additionally, we demonstrate the utility of the variant 17 LbCas12a D156R -VPR for CRISPRa at lower culture temperatures. This is the first 18 demonstration of CRISPRa in filamentous fungi, providing a framework for CRISPR-mediated 19 transcriptional activation of fungal BGCs. 20 activation (CRISPRa)-mediated approach for BGC activation in filamentous fungi (Fig. 1a). In 49 CRISPRa systems, DNase-deactivated RNA-guided CRISPR/dCas ribonucleoprotein 50 complexes linked to activation effectors are targeted to gene regulatory regions to increase 51 gene expression [17][18][19] . Taking advantage of the streamlined CRISPR RNA (crRNA) cloning 52 and multiplexing capabilities, CRISPRa of BGCs has the potential to greatly accelerate fungal 53 genome mining. CRISPRa has already been used to tune the expression of biosynthetic 54 pathways 20,21 , including in ascomycetous yeasts 22,23 . However, to our knowledge, CRISPRa 55has not yet been demonstrated in filamentous fungi. 56In this work, we develop a suite of fungal CRISPRa vectors based on both dLbCas12a from 57Lachnospiraceae bacterium Cas12a (previously known as Cpf1) 19 , and dSpCas9 from 58Streptococcus pyogenes fused to the tripartite VPR activator 18 , and test them in A. nidulans. 59We further explore the application of our CRISPR/dLbCas12a-VPR system for fungal BGC 60 activation as a tool to fuel bioactive molecule discovery. 61 Results 62 Construction and testing of fungal CRISPRa systems 63To develop a CRISPRa system for filamentous fungi, we constructed and tested 64 CRISPR/dLbCas12a-VPR-and CRISPR/dSpCas9-VPR-based systems in the model 65 organism and chassis A. nidulans. To evaluate alternative strategies for expressing either 66 3 dCas effector, we created parent strains with a chromosomally integrated dCas-VPR 67 expression cassette and compared their performance with entirely AMA1-episomally encoded 68 systems. The AMA1 sequence acts as an extrachromosomal vector replicator and confers 69 increased transformation frequency in several filamentous fungi species 24 . AMA1-bearing 70 vectors are found at multiple copies per nucleus although their genetic stability has been 71 reported to be limited under non...

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Section: Resultsmentioning

confidence: 99%

CRISPR-mediated activation of biosynthetic gene clusters for bioactive molecule discovery in filamentous fungi

Roux

Woodcraft

et al. 2020

Preprint

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“…Conventional gene targeting in filamentous fungi requires resistance cassettes with ≥0.5-1 kb flanking homology regions. A major advantage of HR using CRISPR/Cas is that dsDNA repair can also be achieved using short RT homology flanks [23, 31–33]. To test this for B. cinerea , Fen R cassettes with Bos1 homology flanks adjacent to the PAM sequence, ranging from 0 to 60 bp, were generated as RT.…”

Section: Resultsmentioning

confidence: 99%

“…In fungi, advanced CRISPR/Cas systems have been mainly established for Aspergillus spp. [31, 44, 45] and Ustilago maydis [21, 46]. They take advantage of autonomously replicating circular plasmids, namely the Aspergillus -derived AMA1 plasmid and pMS7 in U. maydis , for the delivery of Cas9 and sgRNA.…”

Section: Discussionmentioning

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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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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“…3B). This approach has been applied in other organisms to take advantage of inherent tRNA post-transcriptional processing to express multiple unique sgRNA sequences (42, 44, 45). Combining multiple sgRNAs in an array is particularly useful for R. toruloides, as this minimizes the amount of genetic material that needs to be delivered while maximizing the number of potential gene targets.…”

Section: Resultsmentioning

confidence: 99%