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CRISPR-mediated activation of biosynthetic gene clusters for bioactive molecule discovery in filamentous fungi.
Roux I, Woodcraft C, Hu J, Wolters R, Gilchrist CLM, Chooi YH
ACS Synth Biol. 2020 Jun 11. doi: 10.1021/acssynbio.0c00197. PubMed Article

Plasmids from Article

ID Plasmid Purpose
140193pCRI001-pGEM-LS-Bar-PgpdA-dLbCas12a-VPR-Ttrpc-LSChromosomal integration of PgpdA-dLbCas12a-VPR-TtrpC. Contains 1kb homology arms for Aspergillus nidulans and the bar gene for glufosinate selection. Filamentous fungi integrative vector.
140196pCRI003-pGEM-LS-Bar-PgpdA-dSpCas9-VPR-TtrpC-LSChromosomal integration of PgpdA-dSpCas9-VPR-TtrpC. Contains 1kb homology arms for Aspergillus nidulans and the bar gene for glufosinate selection. Filamentous fungi vector.
140197pCRI004-pYFAC-riboB-PgpdA-dLbCas12a-VPR-TtrpCEpisomal expression of dLbCas12a-VPR. Filamentous fungi vector with AMA1 and riboB selection marker.
140198pCRI005-pYFAC-riboB-PgpdA-dLbCpf1(D156R)-VPR-TtrpCEpisomal expression of dLbCpf1(D156R)-VPR, variant for culture at room temperature. Filamentous fungi vector with AMA1 and riboB selection marker.
140199pCRI006-pYFAC-riboB-PgpdA-dSpCas9-VPR-TtrpCEpisomal expression of dSpCas9-VPR. Filamentous fungi vector with AMA1 and riboB selection marker.
140200pCRI007-pGEM-PgpdA-Cas12aScaffold-BsmbI-TtrpCCloning vector for LbCas12a crRNAs ( Step 1 of the 2-step cloning alternative). Full PgpdA sequence is reconstituted when amplifying the expression cassette for cloning in a fungal vector.
140201pCRI008-PgpdA-Cas12aScaffold-BsmbI-Cas12aScaffold-TrcpTFungal vector for one-step cloning of LbCas12a crRNA arrays and expression. Pgpda and pyroA are BsmbI domesticated.
140202pCRI009-pKW20088-PelcA-mCherry-TtrpC-NotI-PacICRISPRa proof-of-concept test target with fluorescent reporter. PelcA is fused to mCherry, with low basal expression in Aspergillus nidulans. Fungal vector with AMA1 and pyrG selection marker.
140203pCRI011-pYFAC-pyroA-PgpdA-4xcrRNAarrayelcA-TtrpCFungal vector to express an LbCas12a crRNA array targeted to Pelca, to be contransformed with pCRI009 containing PelcA-mCherry in a dLbCas12a-VPR strain as proof-of-concept of CRISPRa.
140204pCRI012-pGEM-PacI-PU3-BsmbI-NotIVector for cloning Cas9 sgRNA ( Step 1 of 2-step cloning). Contains AfU3 promoter driven sgRNA expression cassette flanked by PacI and NotI for further cloning in fungal vector.
140205pCRI018-pKW20088-PelcA-mCherry-TtrpC-PU3-E1-Cas9Scaffold-8xTProof-of-concept of CRISPR/dSpCas9-VPR activation, encoding PelcA-mCherry along with the sgRNA E1 expression cassette, targeting PelcA.

Antibodies from Article