Journal: ACS Synthetic Biology

Article Title: Modular Synthetic Biology Toolkit for Filamentous Fungi

doi: 10.1021/acssynbio.1c00260

Figure Lengend Snippet: Genetic Modules and Other Vectors in the Fungal Toolkit for Modular Cloning (FTK) a

Article Snippet: pFTK093 , 171365 , TU (level 1) , sgRNA transcription unit (MoClo lvl1 unit), P-gpdA-HH-sgRNA-HDV-Ttrpc , pICH47761 (lvl1) , pFC334 (Addgene ID 87846), pLM-AMA18.0 dCas9-VPR (Addgene ID 138945) , ( ) .

Techniques: Cloning, Plasmid Preparation, Selection, Marker, Luciferase, Binding Assay, Sequencing, CRISPR

Journal: Cellular and Molecular Life Sciences

Article Title: Targeted CRISPR activation is functional in engineered human pluripotent stem cells but undergoes silencing after differentiation into cardiomyocytes and endothelium

doi: 10.1007/s00018-023-05101-2

Figure Lengend Snippet: Targeting alternative safe harbor loci. a Strategy for CAG-CRISPRa-mCherry and CAG-mCherry cell lines targeting human ROSA ( hROSA26 ) and CLYBL safe harbor sites in WTC11 stem cells. Double stranded breaks were generated at chr3:9396279–9396304 (between exons 1 and 2) for hROSA26 and chr13:99822977–99822980 (between exons 2 and 3) for CLYBL. The following transgenic lines were generated: ( i ) dCas9-VPR-P2A-mCherry driven by the CAG promoter ( ii ) mCherry driven by CAG; all lines also contained neomycin resistance as a selection marker, with expression driven by the endogenous locus. b CAG transcript levels in hiPSCs and hiPSC-CMs ( left ). dCas9-VPR transcripts were further measured in CRISPRa-mCherry cells in undifferentiated and differentiated states ( right ). Expression is shown with respect to HPRT. 2-way ANOVA, followed by post-hoc Tukey-Kramer test, ( hROSA26 cell line : CAG-mCherry vs. CAG-CRISPRa-mCherry, p = 0.007; cell state : hiPSC vs. hiPSC-CM, p = 0.004; cell line:cell state : p = 0.012; CLYBL cell line : CAG-mCherry vs. CAG-CRISPRa-mCherry, p = 0.011; cell state : hiPSC vs. hiPSC-CM, p = 0.202; cell line:cell state : p = 0.244) and t-test were performed to determine statistical significance. n = 4–6 biological replicates (hiPSCs); n = 2–5 independent differentiations (hiPSC-CMs) . c WTC11 hiPSCs were transduced with indicated gRNAs and harvested for gene expression analysis. mRNA expression is normalized to HPRT housekeeping gene and shown with respect to non-targeting control gRNA samples. One-way ANOVA, followed by post-hoc Tukey-Kramer test, was performed to calculate statistical significance. n = 3 (hROSA26) or 5 (CLYBL) biological replicates. All error bars represent SEM

Article Snippet: To generate the donor CRISPR-mCherry plasmids driven by muscle regulatory cassettes, the promoters were assembled with dCas9-VPR-P2A (Addgene #99373) and mCherry (a gift from Jacob Corn, Addgene #102245) and cloned into the pAAV-Neo-CAG donor backbone, replacing the CAG fragment (pAAV-[CK8e or TNT455]-dCas9-VPR-P2A-mCherry).

Techniques: Generated, Transgenic Assay, Selection, Marker, Expressing, Transduction, Gene Expression, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Targeted CRISPR activation is functional in engineered human pluripotent stem cells but undergoes silencing after differentiation into cardiomyocytes and endothelium

doi: 10.1007/s00018-023-05101-2

Figure Lengend Snippet: Utilization of muscle-specific promoters to drive transgene expression in cardiomyocytes. a Schematic of targeting constructs introduced to the AAVS1 safe harbor locus between exons 1 and 2, driven by muscle regulatory cassettes. The following transgenic lines were generated: ( i ) dCas9-VPR-P2A-mCherry driven by the TNT455 or CK8e promoter ( ii ) mCherry driven by TNT455 or CK8e promoter; all lines also contained neomycin resistance as a selection marker, with expression driven by the endogenous locus. b dCas9-VPR RNA expression in hiPSCs and hiPSC-CMs (Day 14) in AAVS1 -targeted cell lines. Gene expression is normalized to HPRT housekeeping gene. A t-test was performed to determine statistical significance. n = 3–4 biological replicates (hiPSCs); n = 4 independent differentiations (hiPSC-CMs) . Error bars represent SEM. c mCherry expression analysis in Day 14 hiPSC-CMs by flow cytometry. Gating was determined based on age-matched unedited wild type WTC11 hiPSC-CMs. Percent cTNT positive cells are indicated for each differentiation. d mCherry expression from TNT455-regulated cassettes with and without the inclusion of dCas9-VPR as part of the cDNA in Day 14 WTC11 hiPSC-CMs. Scale bar: 200 μm

Article Snippet: To generate the donor CRISPR-mCherry plasmids driven by muscle regulatory cassettes, the promoters were assembled with dCas9-VPR-P2A (Addgene #99373) and mCherry (a gift from Jacob Corn, Addgene #102245) and cloned into the pAAV-Neo-CAG donor backbone, replacing the CAG fragment (pAAV-[CK8e or TNT455]-dCas9-VPR-P2A-mCherry).

Techniques: Expressing, Construct, Transgenic Assay, Generated, Selection, Marker, RNA Expression, Gene Expression, Flow Cytometry

Journal: Cellular and Molecular Life Sciences

Article Title: Targeted CRISPR activation is functional in engineered human pluripotent stem cells but undergoes silencing after differentiation into cardiomyocytes and endothelium

doi: 10.1007/s00018-023-05101-2

Figure Lengend Snippet: Differentiation from mesoderm lineage. WTC11 AAVS1 -CAG-CRISPRa hiPSCs were differentiated into hiPSC-CMs and cardiogenic endothelial cells (iPSC-Endo). Cells were harvested across different time points during differentiation and mature CAG transcript expression was measured. Days of differentiations are indicated (Day 0- primed stem cell; Day 2- mesoderm; Day 5- progenitor; Day 12- cardiomyocyte or endothelial cell). mRNA expression of transcript measured at CAG promoter ( a ) and at dCas9-VPR ( b ) is normalized to HPRT. c CAG-mediated expression measured in AAVS1 -CAG-CRISPRa and CLYBL -CAG-CRISPRa-mCherry cell lines in undifferentiated and Day 14 hiPSC-Endo. A t-test was performed to determine statistical significance. n = 3 biological replicates (hiPSCs); n = 2–4 independent differentiations (hiPSC-CMs); n = 3–4 independent differentiations (hiPSC-Endos) . All error bars represent SEM

Article Snippet: To generate the donor CRISPR-mCherry plasmids driven by muscle regulatory cassettes, the promoters were assembled with dCas9-VPR-P2A (Addgene #99373) and mCherry (a gift from Jacob Corn, Addgene #102245) and cloned into the pAAV-Neo-CAG donor backbone, replacing the CAG fragment (pAAV-[CK8e or TNT455]-dCas9-VPR-P2A-mCherry).

Techniques: Expressing

Journal: Cellular and Molecular Life Sciences

Article Title: Targeted CRISPR activation is functional in engineered human pluripotent stem cells but undergoes silencing after differentiation into cardiomyocytes and endothelium

doi: 10.1007/s00018-023-05101-2

Figure Lengend Snippet: DNA methylation analysis of promoters. a Schematic of methylation-specific PCR (MSP) assay. Genomic DNA is subjected to bisulfite conversion. Converted DNA is used as input for PCR with primers specific for either methylated or unmethylated cytosines for the same site, and subsequently assessed for the presence or absence of product indicating the methylation status of the target region. Locations of MSP primer target sites measured for the CAG promoter are indicated. b WTC11 hiPSC lines, with genes driven by CAG that had been targeted to either the AAVS1 , hROSA26 , or CLYBL genomic loci, were subjected to bisulfite conversion, followed by PCR using primers targeting methylated (M) or unmethylated (U) DNA. Representative gel images are shown for analysis of the hypermethylated region of the CAG promoter. c MSP analysis of differentially methylated region of CAG promoter, using primers targeting methylated DNA, in CLYBL -targeted cell lines. d Genomic DNA from hiPSCs and hiPSC-CMs (Day 14) in CLYBL -targeted cell lines were subjected to methylation sensitive HpaII digestion. Quantitative PCR measures fraction of methylated DNA in the intronic region of the promoter. n = 1–4 biological replicates. 2-way ANOVA, followed by post-hoc Tukey-Kramer test, (Primer Set 1: CLYBL -CAG-mCherry vs. CLYBL -CAG-CRISPRa-mCherry, p = 0.0413; cell state : hiPSC vs. hiPSC-CM, p = 0.304; cell line:cell state : p = 0.772; Primer Set 2: CLYBL -CAG-mCherry vs. CLYBL -CAG-CRISPRa-mCherry, p = 0.037; cell state : hiPSC vs. hiPSC-CM, p = 0.650; cell line:cell state : p = 0.996). e Bisulfite PCR was performed to amplify the CAG intronic sequence. Sanger sequencing was used to assess and quantify percent methylation at individual CG sites within amplicon, spanning 33 CpGs. Plot shows percent methylation of CpGs in sampled PCR products from CLYBL cell lines. x-axis indicates CpG position within the PCR amplicon. f hiPSCs were treated with indicated concentrations of 5-azacytidine (5aza) for 24 h and harvested for mRNA expression analysis of CAG or dCas9-VPR by quantitative rt-PCR. Expression is quantified with respect to HPRT. One-way ANOVA, followed by post-hoc Tukey-Kramer test, was performed to calculate statistical significance. n = 2–7 biological replicates . g Day 16 hiPSC-CMs were treated with 5µM 5aza for 96 h and harvested for CAG transcript expression analysis by quantitative rt-PCR. Expression is quantified with respect to HPRT. A t-test was performed to determine statistical significance. All error bars represent SEM

Article Snippet: To generate the donor CRISPR-mCherry plasmids driven by muscle regulatory cassettes, the promoters were assembled with dCas9-VPR-P2A (Addgene #99373) and mCherry (a gift from Jacob Corn, Addgene #102245) and cloned into the pAAV-Neo-CAG donor backbone, replacing the CAG fragment (pAAV-[CK8e or TNT455]-dCas9-VPR-P2A-mCherry).

Techniques: DNA Methylation Assay, Methylation, MSP Assay, Real-time Polymerase Chain Reaction, Sequencing, Amplification, Expressing, Quantitative RT-PCR

Journal: Biotechnology and bioengineering

Article Title: Awakening dormant glycosyltransferases in CHO cells with CRISPRa

doi: 10.1002/bit.27199

Figure Lengend Snippet: A) gRNAs were designed using the CHO-K1 annotation from NCBI. Since experimentally reported TSSs have not been reported for CHO cells, the TSS was defined as the starting base pair of the annotated target genes. Three gRNAs (red rectangles) were designed between 25 bp and 550 bp upstream of the TSS. gRNA positions for each gene targeted in this study are given in Supplementary Table S2. B) A plasmid encoding VPR-dCas9 was transfected along with either one of the gRNA plasmids or a mix of all gRNA plasmids. The gRNAs guide the VPR-dCas9 to a region upstream of the TSS where VPR increases expression of the target gene.

Article Snippet: The construct is here referred to as VPR_dCas9 and available at Addgene (Addgene Plasmid #134601).

Techniques: Plasmid Preparation, Transfection, Expressing

Journal: Biotechnology and bioengineering

Article Title: Awakening dormant glycosyltransferases in CHO cells with CRISPRa

doi: 10.1002/bit.27199

Figure Lengend Snippet: Activation of Mgat3 and St6gal1 via transient expression of gRNA and VPR-dCas9 was measured by qRT-PCR. CHO cells were transfected with individual gRNAs 1–3, or a mix of the 3 gRNAs in three biological replicates. mRNA was harvested 48h after transfection. For controls, cells were transfected with non-targeting gRNA and VPR-dCas9 (NT-VPR-dCas9) or non-targeting gRNA (NT). Relative expression is calculated with respect to the NT samples. Error bars represent the 95% percentile confidence interval as calculated by the Relative Quantification module of Thermo Fisher Connect (see Methods). Relative expression of Mgat3 and St6gal1 with respect to only the housekeeping genes is shown in Supplementary Figure S2.

Article Snippet: The construct is here referred to as VPR_dCas9 and available at Addgene (Addgene Plasmid #134601).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Transfection