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Synthetic BiologyACS Synthetic Biology
Peer-reviewed journal article

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

Indra RouxiD, Clara WoodcraftiD, Jinyu HuiD, Rebecca WoltersiD, Cameron L. M. GilchristiD, Yit‐Heng ChooiiD

Abstract

Accessing the full biosynthetic potential encoded in the genomes of fungi is limited by the low expression of most biosynthetic gene clusters (BGCs) under common laboratory culture conditions. CRISPR-mediated transcriptional activation (CRISPRa) of fungal BGC could accelerate genomics-driven bioactive secondary metabolite discovery. In this work, we established the first CRISPRa system for filamentous fungi. First, we constructed a CRISPR/dLbCas12a-VPR-based system and demonstrated the activation of a fluorescent reporter in Aspergillus nidulans. Then, we targeted the native nonribosomal peptide synthetase-like (NRPS-like) gene micA in both chromosomal and episomal contexts, achieving increased production of the compound microperfuranone. Finally, multi-gene CRISPRa led to the discovery of the mic cluster product as dehydromicroperfuranone. Additionally, we demonstrated the utility of the variant dLbCas12aD156R-VPR for CRISPRa at room temperature culture conditions. Different aspects that influence the efficiency of CRISPRa in fungi were investigated, providing a framework for the further development of fungal artificial transcription factors based on CRISPR/Cas.Competing Interest StatementThe authors have declared no competing interest.View Full Text

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In-depth summary

✦ Multi-agent verified full-text synthesis

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (20 May 2020) · CC BY 4.0

A dLbCas12a-VPR CRISPR activation system works in Aspergillus nidulans, boosting microperfuranone output from the native micA gene and revealing dehydromicroperfuranone as the mic cluster product.

Most fungal biosynthetic gene clusters are expressed too weakly in the lab to yield detectable products, and current activation relies on promoter swaps, transcription factor overexpression, or cloning large clusters into heterologous hosts. This work tests a programmable CRISPR activation route in filamentous fungi, where several genes can be targeted with crRNA arrays.

Key results

  1. ShownA four-crRNA array expressed from the RNA polymerase II promoter PgpdA activated a PelcA-mCherry reporter with dLbCas12a-VPR, whether the effector was chromosomal or episomal. dSpCas9-VPR was weak when chromosomal and stronger when episomal.

    Microscopy against a no-crRNA control; crRNAs targeted 88-327 bp upstream of the start codon. Chromosomal dSpCas9-VPR activation was visible only at prolonged exposure. sgRNAs on a separate AMA1-pyroA vector gave no fluorescence. · Construction and testing of fungal CRISPRa…

  2. ShownActivating chromosomal micA with dLbCas12a-VPR raised microperfuranone titers. Targeting the 5' UTR (MD array) beat the promoter region (MU array), and single crRNAs gave little or no activation, which indicates synergy among crRNAs.

    MD gave up to 0.6 mg/L; MU gave ~4.5-fold increase (up to 0.2 mg/L); MD plus MU gave up to ~0.8 mg/L. Three biological replicates; Welch's t-test with Holm-Sidak correction, corrected P<0.05. · CRISPR/dLbCas12a-VPR mediated activation of micA…; Statistical analysis; Figure legends

  3. ShownCRISPRa also raised production from episomal micA copies, so the effect is not only due to the chromosomal gene. A TSS annotation-blind design (targeting upstream of the start codon) worked at micA.

    Full-length episomal micA promoter strain reached over 4 mg/L with CRISPRa. A shorter episomal micA variant with low basal production showed a ~30-fold increase over control with MD, reaching ~1.5 mg/L. · CRISPR/dLbCas12a-VPR mediated activation of micA…; Figure legends

  4. ShownCo-activating micA with micB and micC (via crRNA array P plus MD) shifted the profile from microperfuranone to three new peaks. These were assigned to dehydromicroperfuranone, a cytochrome P450-derived product, supported by MS/MS, NMR and promoter-replacement controls.

    Peaks I-III showed m/z 265 [M+H]+, 2 Da below compound 1. Array P targeted a 209bp window in a 396 bp bidirectional promoter. NMR in CDCl3 matched literature shifts for compound 2; PalcA-driven micA-B reproduced the profile. · Multi-gene activation uncovered cryptic…; LC-MS/MS analysis; Compound isolation and NMR structural…

  5. ShownThe D156R variant of dLbCas12a restored activation at 25 °C, where the original system failed, but was weaker at 37 °C. It also improved activation at a TTCN PAM array.

    At 25 °C, D156R gave a ~4.5-fold increase in microperfuranone over the no-crRNA control; dLbCas12a-VPR gave none. D156R produced less at 37 °C than the original. TTCN-targeting array improved over the original at 37 °C. · The variant dLbCas12aD156R improves activation…; Figure legends

Methods, data and reproducibility

VPR was fused to catalytically dead LbCas12a or SpCas9, with crRNA arrays delivered from AMA1 episomal vectors in A. nidulans. Activation was read out by mCherry microscopy and LC-DAD-MS metabolite quantification; structures were assigned by LC-MS/MS and NMR.

  • A. nidulans transformants carrying a PelcA-mCherry reporter with dCas12a or dCas9 activators, imaged by fluorescence microscopy · Aspergillus nidulans · n = three biological replicates (individual colonies) per strain
  • A. nidulans micA CRISPRa strains and controls (chromosomal and episomal micA), culture extracts quantified by LC-DAD-MS · Aspergillus nidulans · n = three biological replicates per strain
  • A. nidulans FGSC A4 genome annotation and predicted BGC coordinates for 5' UTR length and PAM frequency analysis · Aspergillus nidulans · FungiDB version 46; JGI MycoCosm
  • LbCas12a and AsCas12a crystal structures aligned to locate the residue homologous to AsCas12a E174 · PDB 5XUS, 5B43
Methods & tools
dLbCas12a-VPR, dSpCas9-VPR, AMA1 episomal vectors, EuPaGDT, CFM-ID, Agilent 1260 LC-DAD with 6130 quadrupole MS, Thermo Fusion Orbitrap LC-MS/MS, NMR (Bruker Avance IIIHD 500/600MHz), GraphPad Prism 8.3.0, PyMOL 1.7.6.3, Matplotlib, FungiDB
Code
https://github.com/gamcil/5_UTR_analysis/
Data & accessions
https://github.com/gamcil/5_UTR_analysis/
Software & environment
GraphPad Prism 8.3.0; PyMOL 1.7.6.3; FungiDB version 46 annotations; analysis code is a Jupyter notebook (Python package versions not stated).
Reproducibility gaps
Plasmid maps are in Addgene, with codes only in supplementary Table S4. Primary quantitative data and P-values are in supplementary files, not the main text.
Limitations (5)
  • Only one native biosynthetic locus (the micA/mic cluster) was targeted, so broader applicability and robust design guidelines for fungal CRISPRa remain to be established. · stated by the authors
  • Targeting the 5' UTR outperformed the promoter region at micA, but this may be a locus-specific effect, so TSS annotation-blind targeting is untested across other genes. · stated by the authors
  • The D156R variant gave lower microperfuranone production than original dLbCas12a-VPR at 37 °C, and efficiency depended on the selection marker used for crRNA vector delivery. · stated by the authors
  • Activation was inferred from fluorescence images and metabolite titers from three biological replicates; no transcript-level measurements are reported, and mCherry results are qualitative. · noted in this summary
  • Peaks I-III interconverted in solvent and were assigned to dehydromicroperfuranone by NMR in chloroform and MS/MS; no bioactivity of the new compound was assessed. · noted in this summary

Who should read:Read this if you want a proof of concept for CRISPR activation of cryptic biosynthetic gene clusters in filamentous fungi, including crRNA array design, temperature and PAM variants.

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