Journal: bioRxiv

Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

doi: 10.1101/231035

Figure Lengend Snippet: ( a ) Schematic of Cas9 fused to the HUH endonuclease PCV with a covalently attached ssODN. ( b ) SDS-PAGE of Cas9 variants reacted with an Alexa 488 fluorescently labelled ssDNA containing the PCV recognition sequence. The top panel is the coomassie stained gel, and the bottom panel is the identical fluorescently imaged gel. PCV is fused to either the carboxyl (Cas9-PCV) or amino (PCV-Cas9) terminus of Cas9. Cas9-PCV(Y96F) represents catalytically inactive PCV(Y96F) fused to Cas9. (C) SDS-PAGE gel shift assay of Cas9 reacting with two ssDNA templates containing the PCV recognition sequence of differing lengths in a 1:1 ssDNA:Cas9 molar ratio.

Article Snippet: Streptococcus pyogenes Cas9 was amplified out of the plasmid pET15_SP-Cas9 (a gift from Niels Geijsen, Addgene plasmid #62731) and inserted in pTD68_SUMO-PCV2 at the BamHI site using Infusion cloning (Clontech) to create C-terminally fused Cas9-PCV.

Techniques: SDS Page, Sequencing, Staining, Electrophoretic Mobility Shift Assay

Journal: bioRxiv

Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

doi: 10.1101/231035

Figure Lengend Snippet: ( a ) Schematic of split luciferase insertion. The C-terminus of NanoLuc nanoluciferase (HiBiT) is encoded on the 200bp ssODN along with the 5’ PCV recognition sequence and targeted to the 3’ end of GAPDH . ( b ) Assaying luminescence using different Cas9 variants when inserting HiBiT into GAPDH in HEK-293T cells. PCV is fused to either the amino (PCV-Cas9) or carboxyl (Cas9-PCV) terminus of Cas9. Transfections were performed with ssODN lacking the PCV recognition sequence (PCV- ssODN) or ssODN containing the PCV recognition sequence (PCV+ ssODN). Units are displayed in relative light units (RLU) normalized to Cas9. ( c ) The calculated fold change from (b) between the PCV-ssODN and PCV+ ssODN is shown for each variant. ( d ) Targeting the GAPDH locus in U2-OS cells. ( e ) Targeting a locus in vinculin in HEK-293T cells using an ssODN containing the PCV recognition sequence. ( f ) Fold change in RLU compared to Cas9 when varying the amount of RNP (equimolar ssODN). All graphs represent data from one of multiple independent experiments exhibiting similar results. Data are shown as mean +/− SD (n=3). Significance calculated using 2-tailed Student’s t-test: ** P < 0.01, *** P < 0.001, ns = no significance (P >0.05).

Article Snippet: Streptococcus pyogenes Cas9 was amplified out of the plasmid pET15_SP-Cas9 (a gift from Niels Geijsen, Addgene plasmid #62731) and inserted in pTD68_SUMO-PCV2 at the BamHI site using Infusion cloning (Clontech) to create C-terminally fused Cas9-PCV.

Techniques: Luciferase, Sequencing, Transfection, Variant Assay

Journal: bioRxiv

Article Title: Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

doi: 10.1101/231035

Figure Lengend Snippet: ( a ) HEK-293T cells stably expressing a mutant mCherry-GFP reporter are edited by HDR through a frameshift correction, restoring mCherry activity. ( b ) Representative microscopy images of fluorescent reporter editing. ( c ) The percent of mCherry positive cells determined by flow cytometry at two different RNP concentrations using an ssODN containing the PCV recognition sequence ( d ) RNP transfections at 3 pmol in the presence or absence of ssODN. Data are shown as mean +/− SD (n=3). For (c) and (d), the statistical significance of %mCherry positive cells between PCV-fusions of Cas9 and Cas9 alone was <0.001, calculated using 2-tailed Student’s t-test.

Article Snippet: Streptococcus pyogenes Cas9 was amplified out of the plasmid pET15_SP-Cas9 (a gift from Niels Geijsen, Addgene plasmid #62731) and inserted in pTD68_SUMO-PCV2 at the BamHI site using Infusion cloning (Clontech) to create C-terminally fused Cas9-PCV.

Techniques: Stable Transfection, Expressing, Mutagenesis, Activity Assay, Microscopy, Flow Cytometry, Sequencing, Transfection

Journal: Cell reports

Article Title: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin-mediated endocytosis

doi: 10.1016/j.celrep.2021.110008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Briefly, a guide RNA (5′-GCAGATGTAGTGTTTCCACA-3′) targeting the open reading frame in the immediate vicinity of the stop codon was cloned into the Cas9 expression vector pX330-U6-Chimeric_BB-CBh-hSpCas9 (gift from Feng Zhang; ), Addgene plasmid #42230).

Techniques: Derivative Assay, Recombinant, Electron Microscopy, Transfection, Expressing, Plasmid Preparation, Software, Cell Analysis, Gas Chromatography, Mass Spectrometry

Journal: Antioxidants

Article Title: Combined Activity of the Redox-Modulating Compound Setanaxib (GKT137831) with Cytotoxic Agents in the Killing of Acute Myeloid Leukemia Cells

doi: 10.3390/antiox11030513

Figure Lengend Snippet: Assessment of a potential mechanism for Setanaxib effects. ( A , B ) Effect of Setanaxib on the proliferation of human AML cells with NOX4 or p22-phox knockout. KO human AML cells were obtained using CRISPR/Cas9 technology. The cell lines stably express Cas9 and were either transduced with control sgRNA (sgLuci, designated WT) or sgRNA targeting NOX4 or p22-phox , respectively (designated KO). Absence of the targeted gene was detected as described in Materials and Methods. The experimental setup for the drug treatments and assay of proliferation were as in A,B. Cells were counted using a hemocytometer at day 8. Mean ± SD of cell treatments of two to three independently sgRNA-transduced cell batches is presented. ( C ) Comparison of synergy of Setanaxib with daunorubicin in Ba/F3-FLT3-ITD cells harboring Cas9 and transduced with sgLuci (WT) or sg Nox4 (KO). Treatments were performed as in and proliferation/viability was assessed by Cell Titer Blue assay. The individual experiments were normalized to DMSO controls. Mean ± SD (with technical triplicates) for cells from three independent transductions with sgRNA is shown. ( D , E ) The 32D-FLT3-ITD cells were subjected to single or combined drug treatments with daunorubicin, diphenyleneiodonium (DPI) or N-acetylcysteine (NAC) as indicated for 72 h, and proliferation/viability was assessed by Cell Titer Blue assay. Three independent experiments (in triplicate) were conducted; error bars represent mean ± SD. Statistical analyses were carried out using two-tailed t -test (n.s.—not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: In brief, MOLM13, MV4-11, Ba/F3 cells, and HEK293 cells (with tet-inducible NOX4) with stable Cas9 expression were generated by transduction with lentiviral particles expressing Cas9 (Streptococcus pyogenes gene in lentiCas9-Blast plasmid, #52962, Addgene Cambridge, MA, USA) and cell selection with blasticidin using standard techniques, and subsequent clonal selection was employed for high Cas9 levels, monitored by immunoblotting.

Techniques: Knock-Out, CRISPR, Stable Transfection, Transduction, Control, Comparison, Two Tailed Test

Journal: Antioxidants

Article Title: Combined Activity of the Redox-Modulating Compound Setanaxib (GKT137831) with Cytotoxic Agents in the Killing of Acute Myeloid Leukemia Cells

doi: 10.3390/antiox11030513

Figure Lengend Snippet: Setanaxib promotes ROS formation elicited by daunorubicin. ( A – C ) MV4-11 cells were treated with the indicated concentrations of the FLT3-ITD inhibitor AC220 (quizartinib, positive control), the general NOX inhibitor diphenyleneiodonium (DPI), or Setanaxib for 4 h in serum-free medium. Thereafter, cells were lysed and lysates subjected to immunoblotting for assessment of pathway activation. Blots were probed with activation-specific antibodies to pFLT3 (pY589/591), pSTAT5 (pY694), pAkt (Ser473), or pErk1/2 (Thr202/Tyr204) antibodies. Subsequently, blots were stripped and comparable loading was validated by reprobing of the membranes with antibodies against FLT3, STAT5, Akt, or Erk as indicated. ( A ) Representative result. ( B , C ) Quantification of three independent experiments for the indicated signaling molecules. Setanaxib (GKT137831) is abbreviated as GKT. Error bars represent mean ± SD. Statistical analyses were carried out with two-tailed t -test. ( D ) MOLM-13 cells were treated with 5 mM NAC (positive control), 500 nM of the general NOX inhibitor diphenyleneiodonium (DPI), 40 nM of the protein kinase inhibitor midostaurin, or 10 or 30 µM Setanaxib for 24 h. Thereafter, cells were stained with H 2 DCFDA for 30 min and ROS levels were quantified on the flow cytometer (MFI, mean fluorescence intensity). Error bars represent mean ± SD, ( n = 3). Statistical analyses were carried out using two-tailed t -test. ( E ) The 32D-FLT3-ITD cells were treated with the indicated concentrations of the drugs for 24 h. Thereafter, cells were stained with ROS Deep Red dye for 30 min and ROS levels were quantified by flow cytometry. The experiment was conducted three times independently. Error bars represent mean ± SD, ( n = 3). Statistical analyses were carried out using two-tailed t -test. ( F ) HEK293 cells with tetracycline (tet)-inducible NOX4 overexpression and engineered to constitutively express Cas9 were kept uninduced (−tet) or were induced (+tet) to overexpress NOX4 and were mock-treated with solvent or were treated with Setanaxib as indicated. ROS formation was scored with H 2 DCFDA as in ( D ). Error bars represent mean ± SD, ( n = 3). Statistical analyses were carried out using two-tailed t -test (n.s.—not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: In brief, MOLM13, MV4-11, Ba/F3 cells, and HEK293 cells (with tet-inducible NOX4) with stable Cas9 expression were generated by transduction with lentiviral particles expressing Cas9 (Streptococcus pyogenes gene in lentiCas9-Blast plasmid, #52962, Addgene Cambridge, MA, USA) and cell selection with blasticidin using standard techniques, and subsequent clonal selection was employed for high Cas9 levels, monitored by immunoblotting.

Techniques: Positive Control, Western Blot, Activation Assay, Two Tailed Test, Staining, Flow Cytometry, Fluorescence, Over Expression, Solvent