Journal: Haematologica

Article Title: Epigenetic dysregulation of eukaryotic initiation factor 3 subunit E (eIF3E) by lysine methyltransferase REIIBP confers a pro-inflammatory phenotype in t(4;14) myeloma

doi: 10.3324/haematol.2023.283467

Figure Lengend Snippet: RNA-binding protein eIF3E was dysregulated by histone methylation and participates in REIIBP oncognesis. (A) Integrative Genomics Viewer (IGV) browser of representative gene tracks from biological triplicates of eIF3E were shown for H3K4me3 and H3K27me3 histone marks in RPMI8226-vector control (VCon) and RPMI8226-REIIBP cells. CRISPR/Cas9-mediated knockdown of H3K4me3 peak using 3 different single guide RNA (sgRNA) in RPMI8226-REIIBP cells with the positions of the sgRNA indicated. β-actin is the loading control. N=2, independent repeats, representative western blots (WB) shown. (B) Left panel: the global protein synthesis rate was determined by puromycin labeling coupled with immunoblot using antibody against puromycin (12D10). Lanes 1 and 2 are mock treated, lanes 3 and 4 are labeled with 10 µg/mL of puromycin for 10 minutes and lanes 5 and 6 are labeled with puromycin and treated with 100 µM cycloheximide (CHX) for 10 minutes. Right panel: O-propargyl-puromycin (OPP)-labeling coupled with immunofluorescence in RPMI8226-VCon and RPMI8226-REIIBP. Newly synthesized proteins were stained with Alexa Flour 488 (green) and nucleus stained with DAPI (blue). Image is representative field. Scale bar, 100 µm. (C) RNA immunoprecipitation (RNA IP) was performed with eIF3E antibodies or immunoglobuolin (Ig)G control, and binding with TLR7 or BTK mRNA was determined using quantitative real time polymerase chain reaction. Asterisks represent significant differences (* P <0.05; *** P <0.001, Student’s t test). (D) The % transcript distribution of TLR7 mRNA was determined in the non-translating, 80S, low polysome and high polysome after polysome profiling comparing between RPMI8226-VCon and RPMI8226-REIIBP; 56.2% of TLR7 mRNA were associated with polysomes as compared to 64.4% in REIIBP, indicating a higher translation efficiency in REIIBP cells.

Article Snippet: Two single guide RNA (sgRNA) targeting the coding region of eIF3E were cloned into vector backbone pRP(CRISPR)-Puro-hCas9-U6 and three sgRNA targeting its H3K4me3 TSS peak (VectorBuilder, USA).

Techniques: RNA Binding Assay, Methylation, Plasmid Preparation, Control, CRISPR, Knockdown, Western Blot, Labeling, Immunofluorescence, Synthesized, Staining, RNA Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction

Journal: BMC Biology

Article Title: Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library

doi: 10.1186/s12915-020-00905-1

Figure Lengend Snippet: Empirical design of the HD CRISPR library. Schematic illustration of the HD CRISPR library design process. sgRNA sequences that were previously used in negative selection screens contained in the GenomeCRISPR database were annotated with information on rationally selected sequence and phenotype features. All negative selection screens were reanalyzed with BAGEL. High-quality experiments were selected based on how well reference core and nonessential gene sets could be separated in those screens. sgRNAs with high on-target activity were then determined as sequences that both target an essential gene (as determined by BAGEL) and that rank among the 20% most strongly depleted sequences in the screen. Next, sgRNAs that showed unexpected phenotypes compared to other sgRNAs targeting the same gene were flagged as outliers with potential off-target effects. sgRNA sequences were then selected from the resulting pool of sequences to design a genome-wide library consisting of two mutually exclusive sub-libraries A and B, prioritizing sgRNAs with high on-target and low off-target activity

Article Snippet: Oligos encoding the HD CRISPR library A and B sgRNA sequences and flanking regions were ordered as an oligo pool from Twist Biosciences.

Techniques: CRISPR, Selection, Sequencing, Activity Assay, Genome Wide

Journal: BMC Biology

Article Title: Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library

doi: 10.1186/s12915-020-00905-1

Figure Lengend Snippet: Empirically selected sgRNAs in the HD CRISPR library. a Distribution of log2 fold changes for sgRNAs targeting core essential reference genes. The blue curve represents sequences with strong on-target phenotypes. The red curve represents sgRNAs with weak on-target phenotypes (see “ ”). b Phenotypes of sgRNAs targeting the core essential gene NOP2 for 4 screens performed with the library described in Wang et al. . Compared to other NOP2-targeting sgRNAs, sgRNA 9 showed unexpectedly weak depletion in these experiments and was thus labeled “ineffective”. c Distribution of log2 fold changes for sgRNAs targeting nonessential reference genes. The blue curve represents sequences with low off-target activity. The red curve represents sgRNAs that led to unexpectedly strong phenotypes. d Phenotypes of sgRNAs targeting the nonessential gene KRT35 in 4 screens performed with the library described in Wang et al. . Unlike other KRT35-targeting sgRNAs, sgRNA 8 consistently displayed toxic phenotypes in these experiments and was therefore marked as “toxic”. e Percentage of sgRNA sequences in sub-libraries A (left) and B (right) that could be selected based on empirical evidence from published screening experiments. Empirical essential sgRNAs were selected based on inducing a viability phenotype, empirical nonessential sgRNAs based on the absence of a toxic phenotype. f – h Calculated sequence scores applying either the rule set 2 , the DeepHF or the Hart et al. algorithms. Score performance of the HD CRISPR sub-libraries A and B was benchmarked against the libraries whose design is based on respective scores (Brunello for rule set 2, TKOv3 for Hart et al.) if available as well as the GeCKOv2 library and a random sample of sgRNAs from published libraries. The DeepHF score was used as an independent measure none of the investigated libraries was designed on. i – k Comparison of sgRNA scores for empirically and de novo-designed sgRNAs within the HD CRISPR sub-libraries

Article Snippet: Oligos encoding the HD CRISPR library A and B sgRNA sequences and flanking regions were ordered as an oligo pool from Twist Biosciences.

Techniques: CRISPR, Labeling, Activity Assay, Sequencing