coding sequence targeting guide rna  (Synthego Inc)

Journal: Blood Advances

Article Title: Repression of TRIM13 by chromatin assembly factor CHAF1B is critical for AML development

doi: 10.1182/bloodadvances.2022009438

Figure Lengend Snippet: CHAF1B upregulation and TRIM13 downregulation are associated with AML progression. (A-B) Schematic representation of Chaf1b -floxed allele in mouse MLL-AF9 leukemic cells and confirmation of Chaf1b deletion after Cre activation via quantitative polymerase chain reaction (qPCR) (A) and western blot (B). Value shown is mean ± standard deviation (SD) of n = 3 independent experiments. (C) Western blot at 24 and 48 hours after Cre induction in MLL-AF9 leukemic cells. (D) Survival of patients with AML with high or low TRIM13 expression in the Cancer Genome Atlas (TCGA). P value shown. (E) Expression of TRIM13 throughout human hematopoiesis and leukemia from BloodSpot. (F) Scatterplot of expression comparing CHAF1B and TRIM13 in TCGA. The solid red line represents best fit with ± SD (dashed red lines). Line equation, P value, and R 2 value listed. (G) Immunohistochemistry (IHC) in BM biopsies for CHAF1B (top), TRIM13 (middle), and hematoxylin and eosin (H&E) staining of biopsies of healthy BM and from 3 matched patients with initial/relapse or initial/remission AML. Scale bar, 20 μm; IHC and H&E imaged from the same section, if possible. (H) Western blot confirming TRIM13 short hairpin RNA (shRNA) efficacy in mobilized CD34 + PBSCs. Results shown are representative of 3 independent assays. (I) Western blot confirming the CHAF1B overexpression efficacy. Results shown are representative of 3 independent assays. (J) Colony assay in PBSCs expressing indicated viral constructs. Results shown are mean ± SD, dots indicate individual replicates. ∗ P < .05, as determined using 1-way analysis of variance (ANOVA) (J). SCRsh, scrambled control short hairpin; Veh, vehicle.

Article Snippet: Complete single guide RNA targeting early in the coding sequence of TRIM13 were ordered from Synthego.

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation, Expressing, Immunohistochemistry, Staining, shRNA, Over Expression, Colony Assay, Construct, Control

Journal: Blood Advances

Article Title: Repression of TRIM13 by chromatin assembly factor CHAF1B is critical for AML development

doi: 10.1182/bloodadvances.2022009438

Figure Lengend Snippet: CHAF1B represses TRIM13 expression by binding to its promoter. (A) CHAF1B chromatin immunoprecipitation sequencing comparing the occupancies in MOLM13, MV4;11, K562, and U937 AML cell lines. (B) Western blot at indicated timepoints after CHAF1B knockdown in U937 cells. (C,F) Taqman QPCR measuring TRIM13 expression after CHAF1B knockdown. Values shown are mean ± SD relative to SCRsh. (D) Taqman QPCR of TRIM13 expression after CHAF1B overexpression. Values shown are mean ± SD relative to those of nondoxycycline (Dox) control. (E,G) Taqman QPCR measuring TRIM13 expression after CHAF1BRRAA overexpression. Values shown are mean ± SD relative to those of non-Dox control. (H) Cell cycle assay in U937 cells expressing indicated CHAF1Bsh or CHAF1B complementary DNA for the indicated times. (± SD is omitted for simplicity) Bolded conditions are significantly different from that of control. (I) Schematic design of luciferase assay. (J) Western blot of dox-inducible CAF1 or empty vector in indicated cell lines. (K) Luciferase assay normalized for Renilla expression. Values shown are mean values from 3 biological replicates experiments. (H). ∗ P < .05, as determined using one-way ANOVA with Bonferroni post hoc test (C-H) or t test with Welch correction (K).

Article Snippet: Complete single guide RNA targeting early in the coding sequence of TRIM13 were ordered from Synthego.

Techniques: Expressing, Binding Assay, ChIP-sequencing, Western Blot, Knockdown, Over Expression, Control, Cell Cycle Assay, Luciferase, Plasmid Preparation

Journal: Blood Advances

Article Title: Repression of TRIM13 by chromatin assembly factor CHAF1B is critical for AML development

doi: 10.1182/bloodadvances.2022009438

Figure Lengend Snippet: TRIM13 overexpression represses leukemogenesis. (A) Western blot confirmation of TRIM13 overexpression or knockdown in AML cell lines. (B) In vitro suspension culture competitive growth assay. Values shown are mean of 3 independent replicates. (C) Colony formation assay in indicated AML cell lines expressing empty vector or TRIM13 overexpression. (D) Cell death assay measuring annexin V and propidium iodide staining in AML cell lines expressing empty vector of TRIM13 overexpression, taken at day 5. (E) Western blot confirmation of CHAF1B knockdown by shRNA. (F) CD14 surface marker expression on U937 cells 72-hours after CHAF1B knockdown. (G) CD14 surface marker expression on U937 and MOLM13 AML cell lines 5-days after TRIM13 overexpression. Red star indicates significant increase in CD14 expression when compared with empty vector. (H) Western blot confirmation of TRIM13 overexpression in sample of patients with AML. (I) Colony formation assay in indicated sample of patients with AML expressing empty vector or TRIM13 overexpression. (J) Schematic for xenograft transplantation of AML cell lines into NSG mice. (K) Western blot confirmation of TRIM13 overexpression in MOLM13 AML cell line. (L) Percent human CD45+ mononuclear cells in BM aspirates 26 days after transplantation. Each dot represents an individual mouse (n = 10), and line indicates mean. (M) Survival curve of recipient mice receiving TRIM13 overexpressing or control AML cell lines (n = 10 mice). ∗ P < .05, as determined using nonparametric t test with Welch correction (C,D,L), one-way ANOVA (I), and log-rank test (M). Data shown are representative of 3 independent assays (A,E-H,K) and the mean of 3 biological replicates (B) with SD (C-D). FACS, fluorescence-activated cell sorting.

Article Snippet: Complete single guide RNA targeting early in the coding sequence of TRIM13 were ordered from Synthego.

Techniques: Over Expression, Western Blot, Knockdown, In Vitro, Suspension, Growth Assay, Colony Assay, Expressing, Plasmid Preparation, Staining, shRNA, Marker, Transplantation Assay, Control, Fluorescence, FACS

Journal: Blood Advances

Article Title: Repression of TRIM13 by chromatin assembly factor CHAF1B is critical for AML development

doi: 10.1182/bloodadvances.2022009438

Figure Lengend Snippet: TRIM13 repression promotes leukemogenesis. (A) Western blot confirmation of TRIM13 knockdown in PDX 16 to 35 before transplant. (B) Percent human CD45+ mononuclear cells in BM aspirates on indicated days after transplantation. Each dot represents an individual mouse (n = 10), middle line represents mean, box represents SD, and whiskers represent minimum-maximum range. Individual points represent individual mice (n = 10). (C) Survival of recipient mice after transplantation of 1 × 10 6 live AML16 to AML35 cells. (D) Western blot confirmation of TRIM13 knockdown in AML17 to AML129. (E) Colony forming assay in indicated PDXs expressing TRIM13sh2 (T13sh2). (F) Schematic diagram of RING domain deletion in TRIM13 by CRISPR. (G) Western blot confirmation of deletion clones in AML cell lines. ∗ indicates full-length TRIM13. (H) Colony assay in indicated AML cell lines comparing CTRLsg with T13RINGdel cells. (I) Cell death assay at steady state in T13RINGdel clones compared with CTRLsg. Red star indicates significant difference in AnnexinV/propidium iodide between conditions, black start indicates significant differences in AnnexinV between conditions. (J) Cell surface CD14 expression at steady state in AML cell lines. Unless otherwise noted, ∗ P < .05, as determined using t test with Welch correction (B), one-way ANOVA (E,H-I), or log-rank (C). All experiments shown are representative of 3 biological replicates (A,D,G,J), mean + SD of 3 biological replicates (E,H,I), or from 10 mice (B-C). CTRLsg, cells electroporated with a nontargeting control single guide RNA.

Article Snippet: Complete single guide RNA targeting early in the coding sequence of TRIM13 were ordered from Synthego.

Techniques: Western Blot, Knockdown, Transplantation Assay, Expressing, CRISPR, Clone Assay, Colony Assay, Control

Journal: Blood Advances

Article Title: Repression of TRIM13 by chromatin assembly factor CHAF1B is critical for AML development

doi: 10.1182/bloodadvances.2022009438

Figure Lengend Snippet: TRIM13 nuclear localization is necessary for its antileukemic function. (A) Confirmation of TRIM13 antibody specificity (histogram = TRIM13 staining intensity vs frequency). Result shown is quantified from 10 000 U937 cells expressing either SCRsh or TRIM13sh2. (B) Representative images of TRIM13 localization (top image, nuclear localized; bottom image, nonnuclear localized) in U937 AML cell line. Scale bar, 7 μm; cell number (top left). (C-E) Pixel analysis of 10 000 cells: (C) U937 T13RINGdel vs T13WT, (D) AML cell lines, (E) CD34 + PBSCs and PDXs; TRIM13 localization compared with DNA localization (FxCycle Violet). Higher similarity score means TRIM13 and DNA images are more similar. Nuclear localization indicated by gray shading. (F) Representative images of U937 cells expressing either HA-TRIM13 (top) or HA-NES-TRIM13 (bottom). Scale bar, 7 μm; cell number (top left). (G) Pixel analysis of 10 000 U937 cells expressing HA-TRIM13 or HA-NES-TRIM13. Gray shading indicates nuclear localization. (H) Colony forming unit assay in AML cell lines expressing empty vector, HA-TRIM13, or HA-NES-TRIM13. (I) Pixel analysis of 10 000 CD34 + PBSCs or PDXs cells comparing TRIM13 staining intensity to similarity to DNA stain (FxCycle). (J) Pixel analysis of 10 000 CD34 + PBSCs or PDXs cells showing the similarity between TRIM13 staining intensity and DNA staining (FxCycle) intensity. ∗ P < .05, determined using one-way ANOVA, compared with empty vector (H). Results shown are mean ± SD from 3 biological replicates (H), histogram from 10 000 cells (A,C,D,E,G), or representative of 3 biological replicates (I,J).

Article Snippet: Complete single guide RNA targeting early in the coding sequence of TRIM13 were ordered from Synthego.

Techniques: Staining, Expressing, Colony-forming Unit Assay, Plasmid Preparation

Journal: Blood Advances

Article Title: Repression of TRIM13 by chromatin assembly factor CHAF1B is critical for AML development

doi: 10.1182/bloodadvances.2022009438

Figure Lengend Snippet: TRIM13 stabilizes proteins associated with cell cycle. (A) Volcano plot comparing identified protein expression from T13WT with T13RINGdel in nuclear or cytoplasmic extracts. Results are calculated from 3 independent submissions. Red shaded region indicates proteins significantly upregulated ( P < .05) in T13RINGdel compared with T13WT. (B) Volcano plot comparing differentially ubiquitinated peptides in T13RINGdel vs T13WT U937 cells. Blue shading indicates ubiquitinated peptides significantly lost in T13RINGdel ( P < .05). (C) Venn diagram comparing differentially ubiquitinated peptides lost in T13RINGdel with proteins significantly upregulated ( P < .05; more than twofold change in peptide spectrum match, or PSMs). (D) Gene ontology (GO) analysis of proteins with a ubiquitination site lost in T13RINGdel U937 cells. (E) Cell cycle (EdU vs FxCycle) analysis in multiple T13RINGdel clones and TRIM13 overexpression on day 5 after infection in each AML cell line. Bars indicate mean ± SD, and ∗ P < .05 for each specific cell cycle phase within each cell type compared with CTRLsg, as determined using one-way ANOVA on 3 independent biological replicates. (F) Representative imaging cytometry of CCNA1 and TRIM13 localization through cell cycle in MA9.NRAS. (G) Similarity of CCNA1/TRIM13 through cell cycle. Red dashed line is line of best fit by linear regression. Nonzero slope calculated by linear regression modeling and P value listed. Blue shading indicates more similar TRIM13/CCNA1 than dissimilar, and cell cycle stages listed (G0/G1, S, G2, and M) as determined by DNA content and nuclear morphology. (H) Western blot analysis of PBSC expressing TRIM13sh 72-hours after infection and U937 T13RINGdel AML cell line at steady state. (I) Western blot analysis of T13WT vs T13RINGdel U937 cells expressing a dox-inducible CCNA1 at indicated time points after dox induction. Results shown are representative of 2 biological replicates. (J) Colony assay of indicated genotypes of U937 cells expressing either empty vector or CCNA1 in the presence of dox. Results shown are mean ± SD from 4 biological replicates, dots indicate individual replicates. ∗ P < .05 between noted groups (E,H), as determined using one-way ANOVA with Bonferroni post hoc test (H).

Article Snippet: Complete single guide RNA targeting early in the coding sequence of TRIM13 were ordered from Synthego.

Techniques: Expressing, Ubiquitin Proteomics, Clone Assay, Over Expression, Infection, Imaging, Cytometry, Western Blot, Colony Assay, Plasmid Preparation

Journal: Blood Advances

Article Title: Repression of TRIM13 by chromatin assembly factor CHAF1B is critical for AML development

doi: 10.1182/bloodadvances.2022009438

Figure Lengend Snippet: TRIM13 stabilizes CCNA1 through ubiquitination. (A-B) Coimmunoprecipitation (Co-IP) of indicated factors 48 hours after transfection in 293T cells. (C) Co-IP of indicated TRIM13 domain mutants 48 hours after transfection in 293T cells. (D) Denaturing IP for FLAG-CCNA1 in 293T cells expressing TRIM13-V5 and indicated HA-tagged R-K ubiquitin. (E) Cell-free ubiquitination assay with a library of E2s. Bottom panel is ponceau stain, E1/GST-CCNA1/TRIM13-E2s are indicated. (Top) Blot for ubiquitin. (F) Western blot analysis of indicated genotypes U937 cells after cycloheximide chase. (G) Statistical analysis of CCNA1 half-life replicates. (H-J) Western blot analysis of T13WT and T13RINGdel U937 cells treated with indicated inhibitors: MG132 (H), Bafilomycin (BAF) (I-J), and cyclohexamide (CHX) (J). (K) Statistical analysis of CCNA1 half-life replicates in the presence of BAF. Results shown are representative of 3 biological replicates (A-D,F,H-J), or are mean + SD from 3 biological replicates (G,K). (G,K) Normalized to T13WT CCNA1 Veh level.

Article Snippet: Complete single guide RNA targeting early in the coding sequence of TRIM13 were ordered from Synthego.

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Transfection, Expressing, Staining, Western Blot