Journal: bioRxiv

Article Title: TET2 lesions enhance the aggressiveness of CEBPA- mutant AML by rebalancing GATA2 expression

doi: 10.1101/2023.03.28.534511

Figure Lengend Snippet: ( A ) Schematic representation of generation of Tet2 -knockout clones with CRISPR/Cas9. ( B ) Proliferative outgrowth of Cebpa p30/p30 cells with Tet2 indels. ( C ) Volcano plot depicting differentially expressed genes dependent on the Tet2 mutational status in Cebpa p30/p30 cells (n=5–7 per group). ( D ) Experimental setup for evaluating the effect of Tet2 -deficiency ( Tet2 −/− ) in Cebpa DM AML initiation in vivo . ( E ) Myeloid (Mac1 + ) contribution of donor-derived blood and bone marrow (BM) cells evaluated at 12, 24, and 36 weeks after BM transplantation and Cre-LoxP recombination to generate a Cebpa −/p30 and Tet2 −/− hematopoietic compartment (n=3–6 per genotype and timepoint). ( F ) Survival of lethally irradiated recipient mice after BM transplantation and Cre-LoxP recombination (n=12–14/group). ( G ) Volcano plot depicting differentially expressed genes dependent on Tet2 deficiency status in Cebpa −/p30 leukemic blasts (n=3 per group). ( H ) Frequency of proliferating (Ki67 + ) cells in BM of moribund recipient mice (n=3 per group). **=P<0.01, ***=P<0.001, ****=P<0.0001

Article Snippet: For generation of Tet2 or Gata2 mutated clones, Cebpa p30/p30 cells were electroporated with ribonucleoparticles containing recombinant Cas9 nuclease from Streptococcus pyogenes (Sp) (#1081058, IDT), tracrRNA (#1075927, IDT) and crRNAs (Alt-R® CRISPR-Cas9 crRNA, IDT) targeting Tet2 and Gata2 , respectively. crRNAs were designed using the CHOPCHOP web tool (chopchop.cbu.uib.no) ( Supplemental table 4 ). crRNA and tracrRNA molecules were complexed at room temperature and assembled with recombinant SpCas9 according to manufacturer’s protocols (IDT).

Techniques: Knock-Out, Clone Assay, CRISPR, In Vivo, Derivative Assay, Transplantation Assay, Irradiation

Journal: bioRxiv

Article Title: TET2 lesions enhance the aggressiveness of CEBPA- mutant AML by rebalancing GATA2 expression

doi: 10.1101/2023.03.28.534511

Figure Lengend Snippet: ( A ) Experimental setup for evaluating the effect of Gata2 knockdown, via short hairpin RNA (shRNA) mediated silencing, on Cebpa p30/p30 leukemic cells in a competitive in vivo assay. ( B ) Gata2 mRNA in Cebpa p30/p30 leukemic cells prior to transplantation. ( C ) Representative flow cytometry profiles of input and output of shControl (no knockdown), sh Gata2 A (low knockdown), and sh Gata2 D (high knockdown). ( D ) Competitive advantage of targeting shRNA (GFP + ) vs. non-targeting shRNA (YFP + ) cells in vivo assessed as by flow cytometry (n=3–4 per group). ( E ) Experimental setup for Gata2 CRISPR/Cas9 mutagenesis in Cebpa p30/p30 cells, and outgrowth of heterozygous mutated clones. Percentages of Gata2 mutated clones are indicated. ( F ) Growth curve of Cebpa p30/p30 clones with Gata2 mutation ( Cebpa p30/p30 Gata2 +/MUT ) or wild type Gata2 ( Cebpa p30/p30 Gata2 +/+ ). Red lines mark individual clones. (G) Presence or absence of GATA2 mutations ( GATA2 MUT ) in CEBPA double mutated ( CEBPA DM ) AML cases (n=460) with or without TET2 mutations ( TET2 MUT ) in aggregated data from published cohorts – , , , . *=P<0.05, **=P<0.01

Article Snippet: For generation of Tet2 or Gata2 mutated clones, Cebpa p30/p30 cells were electroporated with ribonucleoparticles containing recombinant Cas9 nuclease from Streptococcus pyogenes (Sp) (#1081058, IDT), tracrRNA (#1075927, IDT) and crRNAs (Alt-R® CRISPR-Cas9 crRNA, IDT) targeting Tet2 and Gata2 , respectively. crRNAs were designed using the CHOPCHOP web tool (chopchop.cbu.uib.no) ( Supplemental table 4 ). crRNA and tracrRNA molecules were complexed at room temperature and assembled with recombinant SpCas9 according to manufacturer’s protocols (IDT).

Techniques: Knockdown, shRNA, In Vivo, Transplantation Assay, Flow Cytometry, CRISPR, Mutagenesis, Clone Assay

Journal: bioRxiv

Article Title: TET2 lesions enhance the aggressiveness of CEBPA- mutant AML by rebalancing GATA2 expression

doi: 10.1101/2023.03.28.534511

Figure Lengend Snippet: ( A ) Gata2 mRNA expression in mouse Cebpa p30/p30 leukemic granulocyte/monocyte progenitors (GMPs) vs normal GMPs and, ( B ) CEBPA binding to the Gata2 distal hematopoietic enhancer ( G2 DHE; −77kb) region, data from Jakobsen et al. (n=2–4 per group). ( C ) Schematic genomic view of the Gata2 distal hemeatopoietic enhancer ( G2 DHE), including tracks from CEBPA and H3K27Ac chromatin immunoprecipitation sequencing (ChIP-seq) in Cebpa p /p30 cells (data from Heyes et al. ), TET2 ChIP-seq in AML-ETO expressing cells (data from Rasmussen et al. ),Targeting of the G2 DHE by dual-( D ) or single-( E ) guided CRISPR-Cas9 in Cebpa p30/p30 cells using indicated sgRNAs (n=3/condition). ( F ) Experimental setup for evaluating the effects of Cebpa knockout on Gata2 V2 mRNA expression and DNA methylation of the CpG island at the promoter of Gata2 V2 in MLL-fusion driven AML ( iMLL-AF9 ). ( G ) Cebpa and ( H ) Gata2 V2 mRNA expression upon induction of Cre-LoxP recombination and, ( I ) DNA methylation of the Gata2 V2 promoter CpG-island (2 biological replicates per genotype). ( J ) Frequency of GATA2 and/or TET2 mutations ( GATA2 MUT and TET2 MUT , respectively) in CEBPA high expressing ( CEBPA HIGH n=45) vs. CEBPA low expressing (CEBPA LOW n=61) AML cases, data from Beat AML cohort . *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001

Article Snippet: For generation of Tet2 or Gata2 mutated clones, Cebpa p30/p30 cells were electroporated with ribonucleoparticles containing recombinant Cas9 nuclease from Streptococcus pyogenes (Sp) (#1081058, IDT), tracrRNA (#1075927, IDT) and crRNAs (Alt-R® CRISPR-Cas9 crRNA, IDT) targeting Tet2 and Gata2 , respectively. crRNAs were designed using the CHOPCHOP web tool (chopchop.cbu.uib.no) ( Supplemental table 4 ). crRNA and tracrRNA molecules were complexed at room temperature and assembled with recombinant SpCas9 according to manufacturer’s protocols (IDT).

Techniques: Expressing, Binding Assay, ChIP-sequencing, CRISPR, Knock-Out, DNA Methylation Assay

Journal: Cell

Article Title: Mitochondrial Sirtuin Network Reveals Dynamic SIRT3-dependent Deacetylation in Response to Membrane Depolarization

doi: 10.1016/j.cell.2016.10.016

Figure Lengend Snippet: (A) SIRT3 deacetylase assays were performed by incubating purified, recombinant SIRT3 (0.5 μg) with acetylated substrate, NAD+ and Pnc1. Deacetylase activity was monitored by measuring nicotinamide production after reaction with ortho-pthalaldehyde. (B) HeLa cells were treated with 10 μM CCCP (+) or DMSO control (−) for 10 min. Then, cells were lysed, and acetyl-proteins were immunoprecipitated using α-AcK antibodies. Immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with α-OGDH, α-SIRT3, and α-GLUD1 antibodies. 1:100 of the lysate was loaded for input controls. (C) SIRT3 knockdown HeLa cells were treated −/+ CCCP and analyzed as described for panel B. (D) Purified HeLa cell mitochondria were treated with 10 μM CCCP or DMSO control for 10 min in K-Pi buffer containing succinate, glutamate and malate. Mitochondria were lysed, separated by SDS-PAGE, and analyzed by Western blotting with α-AcK and α-porin antibodies. Porin was used as loading control. (E–F) Mitochondria isolated from wildtype and SIRT3−/− mouse hearts were treated with indicated concentrations of CCCP or DMSO control for 10 min in K-Pi buffer containing succinate, glutamate and malate. Mitochondria were lysed, separated by SDS-PAGE, and acetylation was assessed as for panel D. (F) Acetylation was quantified using ImageJ. ** indicates p-value < 0.01 (3 biological repeats/genotype). (G) MEFs were treated with 10 μM CCCP, 1 μM of rotenone and antimycin A. Fat oxidation assays were performed in basal growth medium containing labeled palmitic acid for 30 min. Released 3H2O was measured and normalized to protein content. n = 2 experiments. (p<0.01). (H) Representative microscope images of membrane potential in SIRT3 inducible knockdown HeLa cells (shSIRT3) and shRNA scramble control HeLa cells treated with 0.2 μM CCCP for 5 minutes. The TMRM signal was monitored and compared with mitochondrial staining by Mitotracker Green. n=4–5 individual experiments. (I) Representative quantification of depletion and recovery of TMRM signal in SIRT3 scramble shRNA control (blue line) or SIRT3-inducible knockdown (shSIRT3, red line) HeLa cells (from panel H). Pictures were taken every 30 sec, mitochondria were detected by Mitotracker Green, and mean value of TMRM signal was calculated using ImageJ. (J–L) TMRM recovery rates collected from 30–40 cells/group demonstrates a significant difference between SIRT3 scramble shRNA control or shSIRT3 HeLa cells (J); TMRM recovery rates in HeLa cells which were transfected with ATP5O siRNA, and then siRNA resistant variants of wildtype ATP5O H135E ATP5O were overexpressed (K); TMRM recovery in H135E ATP5O Crispr/Cas9 heterozygous cells or control HeLa cells (p=0.03). n=3–5 individual experiments. (M) Model of membrane potential regulation of SIRT3 activity through its binding with ATP synthase. In healthy cells with intact mitochondrial membrane potential, SIRT3 binds to ATP synthase. In conditions of disrupted membrane potential and low matrix pH, SIRT3 dissociates from ATP synthase and binds to other targets, which promote restoration of mitochondrial membrane potential. See Figure S7

Article Snippet: Cas9 Nickase mRNA , TriLink , L-6116.

Techniques: Histone Deacetylase Assay, Purification, Recombinant, Activity Assay, Control, Immunoprecipitation, SDS Page, Western Blot, Knockdown, Isolation, Labeling, Microscopy, Membrane, shRNA, Staining, Transfection, CRISPR, Binding Assay

Journal: Cell

Article Title: Mitochondrial Sirtuin Network Reveals Dynamic SIRT3-dependent Deacetylation in Response to Membrane Depolarization

doi: 10.1016/j.cell.2016.10.016

Figure Lengend Snippet:

Article Snippet: Cas9 Nickase mRNA , TriLink , L-6116.

Techniques: Recombinant, Modification, Sequencing, Extraction, shRNA, Software

Journal: eLife

Article Title: Natural variation in C. elegans arsenic toxicity is explained by differences in branched chain amino acid metabolism

doi: 10.7554/eLife.40260

Figure Lengend Snippet:

Article Snippet: Cas9 protein (IDT, 1074182) was injected at 23 µM.

Techniques: Sequencing, Transfection, Construct, Plasmid Preparation

Journal: Nature Communications

Article Title: Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

doi: 10.1038/s41467-022-30228-4

Figure Lengend Snippet: a Two gRNAs (a, b) targeting slc45a2 exon 1 in zebrafish (top). Experimental setup to analyze CRISPR-Cas9, SpG, and SpRY-mediated mutations in zebrafish by injecting one-cell-stage embryos (bottom). b Phenotypes obtained after the injection of the mRNA–gRNA duplex targeting slc45a2 ( albino ) showing different levels of mosaicism (albino-like (alb-like), severe, mild) compared to the WT. Lateral views (scale bar, 1 mm) and insets of the eyes (scale bar, 0.2 mm) of 48 h post-fertilization (hpf) embryos are shown. c Percentage of albino- phenotypes (panel b) in embryos 48 hpf. ( n ) total number of injected embryos. The results were obtained from at least two independent experiments. d Phenotypic evaluation at different concentrations of gRNAs and mRNAs. Stacked bar plots show the percentage of the phenotypes described in panel b. The results were obtained from at least two independent experiments. e gRNAs were complexed with purified proteins to form RNPs for in vitro and in vivo testing in C. elegans by microinjection . f Sequences of gRNAs targeting dpy-10 with distinct complementarity. RNP combinations comprised of each gRNA and WT SpCas9, SpG, or SpRY were tested in vitro. Top bands show uncleaved PCR product. Lower bands show cleaved products. g The dpy-10 matched and +5 gRNAs were tested for in vivo activity in C. elegans by injecting a single gonad arm. Each dot represents the editing efficiency in each P 0 that produced at least 100 F 1 s. The results were obtained from two independent experiments, with both conditions carried out in parallel injections (Student’s t test p value). h Schematic representation of in vivo experiments in C. elegans using a gtbp-1 :: wrmScarlet reporter by screening for loss of fluorescence. dpy-10 gRNA was used for co-CRISPR. i An anti-wrmScarlet gRNA with NGG PAM was complexed with 1.3 µM of SpCas9, SpG, or SpRY to compare their in vivo efficiencies. In a separate experiment, the SpG-anti-wrmScarlet (NGG) RNP was injected at 8.0 µM. Each dot represents the editing efficiency in each P 0 that produced at least five Dpy or Rol F 1 s. (One-way ANOVA followed by Tukey’s test for multiple comparisons p values).

Article Snippet: The two Cas9 variants: SpG (D1135L/S1136W/G1218K/E1219Q/R1335Q/ T1337R) and SpRY (A61R/L1111R/D1135L/S1136W/G1218K/E1219Q/ N1317R/A1322R/R1333P/R1335Q/T1337R) were cloned into the pET-28b- Cas9-His, with DNA gBlocks (IDT) encoding for the mutated regions (Addgene #179318 and #179320).

Techniques: CRISPR, Injection, Purification, In Vitro, In Vivo, Activity Assay, Produced, Fluorescence