Journal: bioRxiv

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.1101/2022.07.13.499814

Figure Lengend Snippet: a. Schematics of CRISPRi transcription repressor domains and general lentiviral expression construct used for all CRISPRi effectors. b. Experimental design to test effects of stable expression of each CRISPRi effector on growth and transcription in K562 cells. c. Growth defects of effector-expressing cells, measured as the log 2 ratio of mCherry-negative (effector-expressing) to mCherry-positive (not effector-expressing) cells in each well. mCherry levels were measured for 19 days after pooling cells. Data represent mean ± SD from three independent transductions of expression constructs. p -values are from an unpaired two-tailed t-test comparing D19 values for each sample to the D19 value for the “no plasmid” sample. Average percent growth defect per day is the log 2 D19 value divided by the number of days, multiplied by 100 for a percent value. d. Clustered heatmap of correlation of transcript counts from K562 cells expressing indicated CRISPRi effectors or a GFP control. Correlations across samples were calculated using normalized counts (reads per million) for all genes with mean normalized count >1 and then clustered using the Ward variance minimization algorithm implemented in scipy. r 2 is squared Pearson correlation. Data represent three independent transductions of expression constructs. e. Number of differentially expressed genes ( p < 0.05) for cells expressing each effector versus cells expressing GFP only. p -values were calculated using a Wald test and corrected for multiple hypothesis testing as implemented in DeSeq2. See also .

Article Snippet: A modified CROP-seq sgRNA lentiviral expression vector (pJR107) was derived from the parental vector pBA950 ( https://www.addgene.org/122239/ ) by incorporating a GFP fluorescent marker and a UCOE element upstream of the EF1alpha promoter to prevent marker silencing. sgRNA targeting sequences were appended with flanking sequence, BstX1/BlpI overhangs, and PCR adapters.

Techniques: Expressing, Construct, Two Tailed Test, Plasmid Preparation

Journal: bioRxiv

Article Title: Selenocysteine metabolism is a targetable vulnerability in MYCN -amplified cancers

doi: 10.1101/2022.05.17.492172

Figure Lengend Snippet: (A) Clonogenic capacity of SK-N-DZ cells in two different FBS batches (FBS1 and FBS2). Rescue in FBS1 was carried out using liproxistatin-1 (Lip-1, 500 nM), βMe (50 μM), NaSe (50 nM) and selenocysteine (50 nM). (B) Analysis of selenium speciation in FBS lacking growth-supporting capacity of SK-N-DZ cells (FBS1). (C) Dose-dependent toxicity of FIN56 and RSL3 in SK-N-DZ expressing a lentiviral construct expressing Flag- LRP8 (LRP8) or empty vector control (Mock). Viability assays were performed in the two different FBS batches described in ( A ). (D) Dose-dependent toxicity of RSL3 in SK-N-DZ cells overexpressing LRP8 (LRP8-OEx) and co-transfected with siRNA targeting SEPHS2 (SEPHS2 KD ) or non-targeting control. (E) Flow cytometry analysis of C11-BODIPY oxidation in SK-N-DZ LRP8 knockdown cells (LRP8 KD ) or non-targeting controls in the presence and absence of ferrostatin-1 (1 μM). (F) Clonogenic capacity of SK-N-DZ LRP8 knockout cells (LRP8 KO ) expressing a FSP1 overexpression construct (FSP1 OE ) or empty vector control.

Article Snippet: The modified CROP-seq-MS2 plasmid was obtained as following: the lentiviral CROP-seq-opti vector (Addgene plasmid #106280 was a gift from Jay Shendure) was sub-cloned via restriction digest of the plasmid with NsiI-HF (NEB, Cat. No. R3127) and SnaBI (NEB, Cat. No. R0130) and the insertion of a synthetic dsDNA fragment coding for a gRNA scaffold sequence with MS2 stem-loop motifs (manufactured by Synbio Technologies, South Brunswick Township, USA).

Techniques: Expressing, Construct, Plasmid Preparation, Control, Transfection, Flow Cytometry, Knockdown, Knock-Out, Over Expression