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

Abstract

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...

Cre/lox-mediated chromosomal integration of biosynthetic gene clusters for heterologous expression in Aspergillus nidulans

“…We selected as a first landing locus the sterigmatocystin ( stc ) biosynthetic gene cluster boundaries located in A. nidulans chromosome IV, as we previously used this locus for chromosomal expression of other genes. 35 After glufosinate selection of the transformant colonies and PCR verification, the strain A. nidulans landing pad 1 (LP1) was isolated for future tests.…”

CRISPR-Based Transcriptional Activation Tool for Silent Genes in Filamentous Fungi

“…In A. niger, Roux and co-workers observed that dCas9-VPR mediated activation of a mCherry fluorescent reporter fused to the transcriptionally silent Parastagonospora nodorum elcA promoter was stronger with sgRNAs targeting closer to the start codon, in a window of 162-342 bp upstream of the ATG 29 . We target a region 106-170 bp upstream the start codon ATG (32-96 bp upstream the TSS) and observe the highest activation with penDE-sgRNA_c targeting 129 bp upstream the ATG, and the least with penDE-sgRNA_a and _b (not detectable by microcopy) targeting closer to the start codon.…”

“…A potential drawback for using dCas12a in fungi is the low activity at temperatures below 28 °C, while most fungal species grow optimally at temperatures between 25 °C and 30 °C. However Roux and co-workers recently engineered an temperature tolerant Cas12a mutant (dLbCas12a D156R -VPR), which was successfully employed for CRISPRa mediated gene activation in A. nidulans at 25 °C 29 . While dCas12a is an attractive choice when aiming to upregulate multiple genes simultaneously, for single target activation dCas9-VPR is still a good option.…”

“…We got significant upregulation of an entire BGC using a single sgRNA targeting the TF of the BGC. For dLbCas12a based upregulation in A. nidulans (the unmutated dLbCas12a grown at 37 °C) multiple crRNAs were required for gene activation 29 . Another consideration when choosing a system is the different PAM requirement, NGG for (d)Cas9 and TTTN for (d)Cas12a.…”

CRISPR-Cas12a induced DNA double-strand breaks are repaired by locus-dependent and error-prone pathways in a fungal pathogen

“…The basic requirement for CRISPR-Cas genome engineering is a Cas endonuclease protein complexed with a single-guide RNA targeting a genomic region following a protospacer adjacent motif (PAM), such as NGG in the case of the commonly used SpCas9 protein from Streptococcus pyogenes (1,2). Another Cas effector, termed Cas12a (formerly named as Cpf1), is an alternative genome editing tool that has several unique features compared to Cas9 based effectors (6)(7)(8)(9)(10)(11)(12). For instance, Cas12a recognizes a T-rich PAM, which can be better suited for editing some genomic regions (7).…”