Journal: Applied Microbiology and Biotechnology

Article Title: A novel fungal gene regulation system based on inducible VPR-dCas9 and nucleosome map-guided sgRNA positioning

doi: 10.1007/s00253-020-10900-9

Figure Lengend Snippet: Schematic drawing of plasmids encoding the components of the VPR-dCas9 activation system. Selection markers for fungal transformation are depicted in grey, promoters in green and terminators in purple. a Plasmid VPR4 carries the gene for the activator VPR-dCas9 (cyan, yellow) which has a synthetic promoter containing estrogen response elements (EREs) as binding motif for the human estrogen receptor (hER). The terminator of VPR-dCas9 is derived from the glucanase gene of Botrytis cinerea . The hER gene (blue) is transcribed constitutively by the coxA promoter from Aspergillus niger and termination is controlled by the terminator region of the tef1 orthologue of A. niger . The produced hER protein remains transcriptionally inactive unless it is converted to an active form (i.e. homodimer formation) by the addition of estrogens (i.e. DES). The selection marker argB is originated from A. nidulans . b Plasmid sgRNA is the parent vector of all sgRNA-carrying plasmids. It carries the sgRNA scaffold (magenta) which consists of the tracrRNA and HDV ribozyme. Directly upstream of the tracrRNA sequence an Eco91I restriction site is introduced for insertion of the specific gRNA sequences together with the hammerhead ribozyme. After insertion, both elements combined form the functional sgRNA cassette which releases the functional sgRNA upon transcription. The sgRNA cassette is constitutively expressed by the gpdA promoter of A. fumigatus and terminated by the terminator of the tef1 gene of A. fumigatus . The selection marker pyroA originates from A. fumigatus . Detailed information about the plasmids and their construction can be retrieved from section “Plasmid generation”. Figure was generated by SnapGene® software (from GSL Biotech; available at snapgene.com )

Article Snippet: VPR was amplified by primer pair VPR -F/R (template source: pWalium20-10XUAS-3XFLAG- dCas9-VPR (Addgene No.: #78897) (Lin et al. )) while dCas9 was amplified by primer pair dCas9 -1-F/R and dCas9 -2-F/R (template source: pCAG- BirA*-dCas9-eGFP which was kindly provided by the Leonardt laboratory (Schmidtmann et al. )).

Techniques: Activation Assay, Selection, Transformation Assay, Plasmid Preparation, Binding Assay, Derivative Assay, Produced, Marker, Sequencing, Functional Assay, Generated, Software

Journal: Applied Microbiology and Biotechnology

Article Title: A novel fungal gene regulation system based on inducible VPR-dCas9 and nucleosome map-guided sgRNA positioning

doi: 10.1007/s00253-020-10900-9

Figure Lengend Snippet: Targeted activation of the transcription factor gene mdpE via VPR-dCas9 and MdpE-mediated activation of the backbone gene mdpG. a Promoter region of the target gene mdpE together with RNA-sequencing data (grey histograms; Gacek-Matthews et al. ) and nucleosome positioning maps (purple histograms) under nutrient-rich (15/17 h) and nutrient-depleted (48 h) conditions. The nucleosome-free region (NFR) is shown in grey white pattern which is delimited by the nucleosomal borders (set to an offset of 75 base pairs proximal to the dyad axis of the neighbouring nucleosomes). The dyad axes are shown in vertical blue dashed lines and were set at the peak of the nucleosome histogram. Positions of sgRNAs are depicted in red (m1–m4). b Expression of the gene mdpE in the controls (only pVPR4) and strains that additionally carry one or more sgRNAs (m1 | m2 | m3 | m4 | m1&2 | m2&3 | mAll) that target the promoter region of the gene mdpE . c Expression of the backbone gene mdpG in the same samples as shown in b which is caused by the activation of mdpE . Cultures were done in triplicates, and qPCR was done in technical duplicates. A Student’s t test was used to verify the significance of the activation over the controls (i.e. VPR4) (* p < 0.05; ** p < 0.01)

Article Snippet: VPR was amplified by primer pair VPR -F/R (template source: pWalium20-10XUAS-3XFLAG- dCas9-VPR (Addgene No.: #78897) (Lin et al. )) while dCas9 was amplified by primer pair dCas9 -1-F/R and dCas9 -2-F/R (template source: pCAG- BirA*-dCas9-eGFP which was kindly provided by the Leonardt laboratory (Schmidtmann et al. )).

Techniques: Activation Assay, RNA Sequencing, Expressing

Journal: Applied Microbiology and Biotechnology

Article Title: A novel fungal gene regulation system based on inducible VPR-dCas9 and nucleosome map-guided sgRNA positioning

doi: 10.1007/s00253-020-10900-9

Figure Lengend Snippet: Targeting of VPR-dCas9 to a bidirectional promoter and activation of its underlying genes AN8506 and AN8507. a The section shown comprises the begin of both genes AN8506 and AN8507 which share a bidirectional promoter of around 305 base pairs. The histograms show the mRNA profile (grey; Gacek-Matthews et al. ) and the nucleosome positioning (purple) under nutrient-rich (15/17 h) and nutrient-depleted (48 h) conditions. The nucleosome-free region (NFR) is shown in grey white pattern which is delimited by the nucleosomal borders (set to an offset of 75 base pairs proximal to the dyad axis of the neighbouring nucleosomes). The dyad axes are shown in vertical blue dashed lines and were set at the peak of the nucleosome histogram. Positions of sgRNAs are depicted in red (A1 and A2). b Expression data of the activation experiment: Controls: VPR4 (no sgRNA), A1: VPR4-A1-T1 to T3 | A2: VPR4-A2-T1 to T3. Three independent transformants per sgRNA (T1, T2, T3). Cultures were executed in triplicates. qPCR was performed in technical duplicates. Reproducibility was verified by two independent conductions of the experiment. The significance of the activation effect was verified by a Student’s t test (* p < 0.05; ** p < 0.01)

Article Snippet: VPR was amplified by primer pair VPR -F/R (template source: pWalium20-10XUAS-3XFLAG- dCas9-VPR (Addgene No.: #78897) (Lin et al. )) while dCas9 was amplified by primer pair dCas9 -1-F/R and dCas9 -2-F/R (template source: pCAG- BirA*-dCas9-eGFP which was kindly provided by the Leonardt laboratory (Schmidtmann et al. )).

Techniques: Activation Assay, Expressing

Journal: Applied Microbiology and Biotechnology

Article Title: A novel fungal gene regulation system based on inducible VPR-dCas9 and nucleosome map-guided sgRNA positioning

doi: 10.1007/s00253-020-10900-9

Figure Lengend Snippet: VPR-dCas9 activation and experimental design. Green or grey rectangulars represent expressed or not expressed genes respectively. Green arrows indicate active promoters while orange arrows represent promoters poised for activation. a All strains carry plasmid VPR4 (control as well as activation strains). pVPR4 contains the constitutively expressed (P.const; cyan) human estrogen receptor gene (hER) which is inactive in absence of the inducer. Upon induction with DES, hER is activated and able to bind to estrogen response elements (EREs; vertical yellow bars) which are present in the engineered VPR-dCas9 gene promoter. Upon binding, hER facilitates transcription of the gene that codes for VPR-dCas9. b In the control strain (only pVPR4), sgRNAs are not expressed and thus VPR-dCas9 is present in the cell but not targeted to the designated region of interest (horizontal violet bars). The activation strains (pVPR4 and one or more psgRNA with functional sgRNA cassette) constitutively express sgRNA(s) (target sequence in violet) which guide VPR-dCas9 to the region of interest (horizontal violet bars) and facilitate transcription of the targeted gene (i.e. TF). Hence, the activation strain should experience an elevated expression of the targeted gene compared with the control strain, which can only be attributed to the presence of the sgRNA. In one application, the targeted gene could be a transcription factor gene (TF) of a silent BGC and forced expression of this TF gene could subsequently lead to the upregulation of the whole cluster as shown in the figure. In another scenario, several sgRNAs could be targeted to many genes within a predicted cluster and the cognate metabolite(s) could be identified subsequently

Article Snippet: VPR was amplified by primer pair VPR -F/R (template source: pWalium20-10XUAS-3XFLAG- dCas9-VPR (Addgene No.: #78897) (Lin et al. )) while dCas9 was amplified by primer pair dCas9 -1-F/R and dCas9 -2-F/R (template source: pCAG- BirA*-dCas9-eGFP which was kindly provided by the Leonardt laboratory (Schmidtmann et al. )).

Techniques: Activation Assay, Plasmid Preparation, Control, Binding Assay, Functional Assay, Sequencing, Expressing

Journal: Applied Microbiology and Biotechnology

Article Title: A novel fungal gene regulation system based on inducible VPR-dCas9 and nucleosome map-guided sgRNA positioning

doi: 10.1007/s00253-020-10900-9

Figure Lengend Snippet: Overview of the monodictyphenone cluster of A. nidulans . The section spans the whole cluster. Apart from gene annotations (arrows), nucleosome positioning as well as the mRNA profile (Gacek-Matthews et al. ) at nutrient-rich (15/17 h) and nutrient-depleted (48 h) conditions are depicted in purple and grey histograms respectively. White arrows are genes that are not necessary for the synthesis of the BGC products. Grey, orange and ruby arrows are genes involved in the monodictyphenone biosynthesis. The orange arrow is the backbone gene of the cluster which encodes for the non-reduced polyketide synthase MdpG. The ruby arrow is the gene that encodes for the pathway-specific transcription factor MdpE which is the target for the activation by VPR-dCas9

Article Snippet: VPR was amplified by primer pair VPR -F/R (template source: pWalium20-10XUAS-3XFLAG- dCas9-VPR (Addgene No.: #78897) (Lin et al. )) while dCas9 was amplified by primer pair dCas9 -1-F/R and dCas9 -2-F/R (template source: pCAG- BirA*-dCas9-eGFP which was kindly provided by the Leonardt laboratory (Schmidtmann et al. )).

Techniques: Activation Assay

Journal: Applied Microbiology and Biotechnology

Article Title: A novel fungal gene regulation system based on inducible VPR-dCas9 and nucleosome map-guided sgRNA positioning

doi: 10.1007/s00253-020-10900-9

Figure Lengend Snippet: Overview of the predicted cluster AN8504 of A. nidulans . The cluster (as predicted by antiSMASH) begins at gene AN8495 and ends at AN8508. Nucleosome positioning as well as the mRNA profile (Gacek-Matthews et al. ) at nutrient-rich (15/17 h) and nutrient-depleted (48 h) conditions is depicted in purple and grey histograms respectively. The backbone gene (AN8504; orange arrow) shows no transcription at either condition. Two sgRNAs were designed that target VPR-dCas9 to the bidirectional promoter that regulates the genes AN8506 and AN8507 (red frame). AN8506 (ruby arrow) is the putative transcription factor and AN8507 (cyan arrow) is the putative membrane protein. AN8506 shows a low basal transcription level (comparable with 0.32 times of the expression level of the housekeeping gene benA) while AN8507 shows no reads at either condition

Article Snippet: VPR was amplified by primer pair VPR -F/R (template source: pWalium20-10XUAS-3XFLAG- dCas9-VPR (Addgene No.: #78897) (Lin et al. )) while dCas9 was amplified by primer pair dCas9 -1-F/R and dCas9 -2-F/R (template source: pCAG- BirA*-dCas9-eGFP which was kindly provided by the Leonardt laboratory (Schmidtmann et al. )).

Techniques: Membrane, Expressing

Journal: bioRxiv

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

doi: 10.1101/2020.10.13.338012

Figure Lengend Snippet: CRISPRa (dCas9-VPR) based activation of penDE-CP_DsRed . ( a ) Schematic representation of the penDE-CP upstream DsRed . The transcription start site (TSS) is indicated as a black arrow, short lines with letters indicate targeting sites of the sgRNAs. ( b ) Confocal fluorescence microscopy imaging of DsRed targeting CRISPRa strains and controls with no-sgRNA (AMA18.0) and without the penDE-CP_DsRed transcription unit (DS68530). Strains were grown for 5 days in liquid SMP media. Scale bars represent 50 μm. ( c ) Development of DsRed/biomass over time during time window of 0-40 hours cultivation in the BioLector system. Data were obtained from 3 separate experiments, each consisting of 2-3 biological replicates; error bars show the standard deviation.

Article Snippet: CRISPRa vector pLM-AMA18.0 is deposited to AddGene under ID #138945.

Techniques: Activation Assay, Fluorescence, Microscopy, Imaging, Standard Deviation

Journal: Scientific Reports

Article Title: Improved drought stress tolerance in Arabidopsis by CRISPR/dCas9 fusion with a Histone AcetylTransferase

doi: 10.1038/s41598-019-44571-y

Figure Lengend Snippet: Molecular characterization of transgenic A. thaliana dCas9 HAT lines. ( A ) Schematic representation of the construct allowing the selection of A. thaliana dCas9 HAT lines. KanR: the kanamycin resistance gene; mOFP: monomeric orange fluorescent protein; NLS: nuclear localization signal. The white arrows indicate the cauliflower mosaic virus (CaMV) 35 S promoter; the white squares indicate the CaMV poly(A) signal (terminator); the black arrows indicate qPCR oligos ( B ) Fluorescence microscopy imaging of A. thaliana leaves, primordia and roots expressing the mOFP in the nucleus in three dCas9HAT lines compared with Col-0 plants. All confocal images were acquired under identical parameters (excitation: 549 nm/emission: 565 nm). Scale bars in the left inferior corner = 50 μm ( C ) Assessment of dCas9 HAT expression. RT-qPCR was performed in Col-0 plants and in three lines of dCas9 HAT -transformed plants. Transcript levels were normalized against the geometric mean of the transcript levels of the housekeeping genes (GAPDH and Actin2). The mean and standard deviation (SD) were calculated from three independent biological replicates. The calibrator was chosen as the sample with the lowest expression of the transgene (excluding Col-0 plants). Asterisks indicate significant differences between Col-0 plants and the different lines (Wilcoxon test, *P < 0.05).

Article Snippet: The sequence encoding the A. thaliana acetyltransferase domain was gathered from TAIR from the protein HAC1 (AT1G79000, between amino acids (AA) 1119 and 1408), synthesized (EPOCH) and cloned into a vector containing the sequence encoding dCas9 (pAWG-dCas9-VPR, which was a gift from George Church; plasmid #63802; http://n2t.net/addgene:63802 ; RRID: Addgene_63802) .

Techniques: Transgenic Assay, Construct, Selection, Virus, Fluorescence, Microscopy, Imaging, Expressing, Quantitative RT-PCR, Transformation Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Improved drought stress tolerance in Arabidopsis by CRISPR/dCas9 fusion with a Histone AcetylTransferase

doi: 10.1038/s41598-019-44571-y

Figure Lengend Snippet: Challenge of dCas9 HAT in a GUS reporter system. ( A ) Schematic representation of the GmUcesMin promoter with the sgRNA positions (black and white squares). The TATA box is represented by a black square. The black and white curved arrows represent the TSS and ATG codon, respectively. ( B ) Arabidopsis seedlings from stably transformed dCas9 HAT fusions were transiently transformed with GmUcesMin and different combinations of sgRNAs. The results are presented as the mean and SD of 6 independent experiments (n = 20 pooled seedlings per experiment). Asterisks indicate significant difference between the GUS activity for each dCas9 fusion guided by one sgRNA or a combination of two sgRNAs compared to mock controls (Student’s t-test, *P < 0.05). Bars indicate the standard error. ( C ) GUS staining was performed for the GmUcesMin promoter in the same transiently transformed seedlings.

Article Snippet: The sequence encoding the A. thaliana acetyltransferase domain was gathered from TAIR from the protein HAC1 (AT1G79000, between amino acids (AA) 1119 and 1408), synthesized (EPOCH) and cloned into a vector containing the sequence encoding dCas9 (pAWG-dCas9-VPR, which was a gift from George Church; plasmid #63802; http://n2t.net/addgene:63802 ; RRID: Addgene_63802) .

Techniques: Stable Transfection, Transformation Assay, Activity Assay, Staining

Journal: Scientific Reports

Article Title: Improved drought stress tolerance in Arabidopsis by CRISPR/dCas9 fusion with a Histone AcetylTransferase

doi: 10.1038/s41598-019-44571-y

Figure Lengend Snippet: Challenge of dCas9 HAT constructs in the transcriptional regulation of AREB1 by targeting p AREB1 . ( A ) Schematic representation of p AREB1 with the two sgRNAs designated. The TATA box is represented by a black square. ( B ) Relative expression of the AREB1 and ( C ) Phenotypic analysis of dCas9 HAT -sgA. Rosette diameter of three-week-old plants. The results represent the mean of n = 13. dCas9 HAT control plants are represented by a – symbol, and dCas9 HAT- sgA2 plants are represented by a + symbol. Asterisks indicate significant difference between dCas9-sgA lines and dCas9 control lines (Wilcoxon test, *P < 0.05). Bars indicate standard error.

Article Snippet: The sequence encoding the A. thaliana acetyltransferase domain was gathered from TAIR from the protein HAC1 (AT1G79000, between amino acids (AA) 1119 and 1408), synthesized (EPOCH) and cloned into a vector containing the sequence encoding dCas9 (pAWG-dCas9-VPR, which was a gift from George Church; plasmid #63802; http://n2t.net/addgene:63802 ; RRID: Addgene_63802) .

Techniques: Construct, Expressing, Control

Journal: Scientific Reports

Article Title: Improved drought stress tolerance in Arabidopsis by CRISPR/dCas9 fusion with a Histone AcetylTransferase

doi: 10.1038/s41598-019-44571-y

Figure Lengend Snippet: Molecular and physiological analyses of drought stress responses in dCas9 HAT- sgA. Transcript levels of ( A ) AREB1 and ( B ) RD29A in dCas9 HAT and dCas9 HAT -sgA2 plants during drought stress. Expression levels were normalized against the geometric mean of the expression of the housekeeping genes (GAPDH and Actin2). The mean and SD were obtained from three biological replicates. Asterisks indicate significant differences between the control and transformed plants (Wilcoxon test, *P < 0.05). For each gene, the expression level in the dCas9 HAT control was defined as the calibrator (1.0). ( C ) Total chlorophyll content in non-stressed plants 4 h after SDS and after MSDS. The results represent the mean of n = 6. ( D ) Stomatal aperture measurements after 2 h and 4 h of severe stress and 20 days of drought stress. The results are presented as the mean of n = 30. Asterisks indicate significant difference between co-transformed plants and control lines (Student’s t-test, *P < 0.05). The bars indicate standard error. ( E ) Survival rates after 6 h of SDS and 48 h and 96 h of rehydration. The results represent the percentage of surviving plants (n = 20). An asterisk indicates a significant difference between the control dCas9 HAT and dCas9 HAT -sgA2 plants (chi-square test, *P < 0.05). dCas9 HAT control plants are represented by a – symbol, and dCas9 HAT sgA2 plants are represented by a + symbol.

Article Snippet: The sequence encoding the A. thaliana acetyltransferase domain was gathered from TAIR from the protein HAC1 (AT1G79000, between amino acids (AA) 1119 and 1408), synthesized (EPOCH) and cloned into a vector containing the sequence encoding dCas9 (pAWG-dCas9-VPR, which was a gift from George Church; plasmid #63802; http://n2t.net/addgene:63802 ; RRID: Addgene_63802) .

Techniques: Expressing, Control, Transformation Assay

Journal: Scientific Reports

Article Title: Improved drought stress tolerance in Arabidopsis by CRISPR/dCas9 fusion with a Histone AcetylTransferase

doi: 10.1038/s41598-019-44571-y

Figure Lengend Snippet: Schematic illustration of a model for dCas9 HAT function in transcriptional activation of a target gene. Upper: the histone compaction (in orange) induces DNA condensation and limits transcription. Below: the dCas9HAT in complex with a single guide RNA (in dark blue) binds DNA on a target locus. The histone acetyl-transferase (HAT) triggers histone acetylation on the lysine 27 (H3K27ac) and induces local DNA relaxation. The DNA relaxation strengthens the interaction of the transcriptional machinery and /or transcriptional enhancers with the target locus.

Article Snippet: The sequence encoding the A. thaliana acetyltransferase domain was gathered from TAIR from the protein HAC1 (AT1G79000, between amino acids (AA) 1119 and 1408), synthesized (EPOCH) and cloned into a vector containing the sequence encoding dCas9 (pAWG-dCas9-VPR, which was a gift from George Church; plasmid #63802; http://n2t.net/addgene:63802 ; RRID: Addgene_63802) .

Techniques: Activation Assay