Journal: Nature genetics

Article Title: The impact of nonsense-mediated mRNA decay on genetic disease, gene editing and cancer immunotherapy

doi: 10.1038/s41588-019-0517-5

Figure Lengend Snippet: a , a decrease in protein expression due to tiling sgRNAs placed along the length of human and mouse genes (y axis quantifies the sgRNA fold difference between a low-expressing versus high-expressing set of cells) reveals, overall, similar associations with the non-canonical start-proximal NMD rule to the canonical NMD last-exon rule. The CD13 gene demonstrates the effect of the non-canonical long-exon rule. Shaded regions are 95% confidence interval of the loess fit to protein expression. Pearson correlation coefficients and two-sided tests for association were computed by comparing the loess fit to the NMDetective -A NMD efficacy scores. b , evading NMD attenuates the loss of fitness (y axis) due to knockout of essential genes. Data for non-essential genes are in . P values are by Mann-Whitney U test. The knock-out efficiency compares the reduction of sgRNAs in NMD evading regions to the reduction in regions that trigger NMD. c , a ‘saturation genome editing’ CRISPR experiment shows strongly reduced mRNA levels for nonsense mutations in BRCA1, except for those introduced into regions covered by the start-proximal (top) and last-exon NMD evasion rules (bottom panel).

Article Snippet: We therefore used the CRISPR design tools E-CRISP and ‘CRISPRko’ offered by the Genetic Perturbation Platform of the Broad Institute, to design sgRNAs for knock-out experiments of the top 100 most cited genes (from http://doi.org/10.5281/zenodo.1066066 ).

Techniques: Expressing, Knock-Out, MANN-WHITNEY, CRISPR

Journal: Nature genetics

Article Title: The impact of nonsense-mediated mRNA decay on genetic disease, gene editing and cancer immunotherapy

doi: 10.1038/s41588-019-0517-5

Figure Lengend Snippet: a , fitness loss upon targeting a non-essential gene (left) versus an essential gene (right) using a sgRNA directed at gene sections which are covered by various NMD-evasion rules. b - e , distribution of loci targeted by sgRNAs that are NMD-detected or NMD-evading (according to the individual NMD rules) for genome-wide CRISPR libraries ( b , c ) or by sgRNA design tools ( d , e ).

Article Snippet: We therefore used the CRISPR design tools E-CRISP and ‘CRISPRko’ offered by the Genetic Perturbation Platform of the Broad Institute, to design sgRNAs for knock-out experiments of the top 100 most cited genes (from http://doi.org/10.5281/zenodo.1066066 ).

Techniques: Genome Wide, CRISPR

Journal: Scientific Reports

Article Title: Probing the requirement for CD38 in retinoic acid-induced HL-60 cell differentiation with a small molecule dimerizer and genetic knockout

doi: 10.1038/s41598-017-17720-4

Figure Lengend Snippet: CRISPR/Cas9-mediated disruption of CD38 does not affect signalling proteins known to be involved in RA-induced differentiation. Wild-type and CD38 CRISPR cell lines were cultured for 48 h with 1 μM RA as indicated and whole cell lysate was collected. 25 μg of lysate per lane was run. Western blots of PAGE-resolved lysates from wild-type cells and CRISPR 1, 2, and 3 cell lines were probed for the indicated proteins, where GAPDH is a loading control. Membrane images for each protein are cropped to show only the band of interest.

Article Snippet: Lentiviral particles were produced using 2.5 µg pMD2.g (Addgene #12259), 7.5 µg psPAX2 (Addgene #12260), and 10 µg pLentiCRISPR v2 with CRISPR 1, 2, or 3 inserted.

Techniques: CRISPR, Cell Culture, Western Blot

Journal: Stem cell research

Article Title: Generation of a dual edited human induced pluripotent stem cell Myl7-GFP reporter line with inducible CRISPRi/dCas9

doi: 10.1016/j.scr.2022.102754

Figure Lengend Snippet: Generation and characterization of inducible CRISPRi iPSCs.

Article Snippet: Addgene/public access repository recombinant DNA sources’ disclaimers (if applicable) , pAAVS1-NDi-CRISPRi (Gen1) was a gift from Bruce Conklin (Addgene plasmid # 73497; https://n2t.net/addgene:73497 ; RRID: Addgene_73497) Dox-inducible CRISPR interference (CRISPRi) knock-in construct into the AAVS1 locus with mCherry marker..

Techniques:

Journal: Stem cell research

Article Title: Generation of a dual edited human induced pluripotent stem cell Myl7-GFP reporter line with inducible CRISPRi/dCas9

doi: 10.1016/j.scr.2022.102754

Figure Lengend Snippet: Resource Table

Article Snippet: Addgene/public access repository recombinant DNA sources’ disclaimers (if applicable) , pAAVS1-NDi-CRISPRi (Gen1) was a gift from Bruce Conklin (Addgene plasmid # 73497; https://n2t.net/addgene:73497 ; RRID: Addgene_73497) Dox-inducible CRISPR interference (CRISPRi) knock-in construct into the AAVS1 locus with mCherry marker..

Techniques: Cell Culture, Modification, CRISPR, Plasmid Preparation, Sequencing, Activity Assay, In Silico, Expressing, Recombinant, Knock-In, Construct, Marker

Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: (A) A two-week growth CRISPR screen of 150 common Ras-proximal proteins in diploid primary human melanocytes (MC) and 5 MT Ras cell lines. Left heatmap negative selection FDR values (FDR ≤ 0.18) in each cell type by the MAGeCK algorithm (Li et al., 2014). The right heatmap relative enrichment MT vs WT Ras in mass spectrometry data for each Ras isoform; new proteins (red), known proteins (*). Ranked by combined FDR and log2(PSM MT/WT Ras) score. (B) Common Ras-proximal interacting protein-protein network based on candidates with ≥1 database interaction with another common interactor. Large squares are novel and small squares are known interactors. (C) PLA in MT NRAS MM415 melanoma cells with endogenous mTOR and Pan-Ras or Ras:GTP; interaction (red), nuclei (blue). Scale bar, 20 μm. (D) Quantification of PLA analysis in (C). n=8-10 fields/condition. (E) Western blot of HA co-immunoprecipitation of empty vector (EV), FLAG-HA-6xHIS tagged NRASWT or FHH: NRASQ61K with endogenous mTOR, p110α and Raf1 in wild-type RAS CHL-1 cells. (F) Quantification of HA co-IP experiments as in (E). Values are normalized to HA pulldown signal and relative to NRASWT signal, n= 6 (Wilcoxon Signed Rank Test). (G) PLA in genotyped human colorectal adenocarcinomas with endogenous Pan-Ras and mTOR. Scale bar, 20 μm. (H) Quantification of PLA analysis in (G). Each dot represents median signal per patient. n=5 patients/genotype group, ≥7 images analyzed per patient (Mann-Whitney U Test). (I) Microscale thermophoresis with labeled FHH:Raf1RBD (18.2nM) with a titration series of GDP or GTPγs-loaded Ras. The binding curve is negative as the MST signal of the complex is lower than that of Raf1RBD alone. MST-on time of 15s, n = 3 independent replicates. (J) Microscale thermophoresis with labeled FHH:mTORKinaseDomain and FHH:mTORHEAT (23.4nM) with a titration series of GDP or GTPγs-loaded Ras. MST-on time of 2.5s, n ≥ 3 independent replicates. MST is mean ± SD. All other data mean ± SEM; *p< 0.05. See also Figures S3 and S4, Tables S2–4.

Article Snippet: CRISPR Library Screen Prior to infection with the CRISPR library, CHL-1, MM415, MM485, BxPC-3, AsPC1, Caco-2, LS174T, HT-1376, T24, and primary melanocytes we infected with pLEX_Cas9 (pLEX Cas9-FLAG-P2A-blasticidin; Addgene #117987) lentivirus at the highest tolerated dose, as determined by impact on growth compared to uninfected control cells.

Techniques: CRISPR, Selection, Mass Spectrometry, Western Blot, Immunoprecipitation, Plasmid Preparation, Co-Immunoprecipitation Assay, MANN-WHITNEY, Microscale Thermophoresis, Labeling, Titration, Binding Assay

Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CRISPR Library Screen Prior to infection with the CRISPR library, CHL-1, MM415, MM485, BxPC-3, AsPC1, Caco-2, LS174T, HT-1376, T24, and primary melanocytes we infected with pLEX_Cas9 (pLEX Cas9-FLAG-P2A-blasticidin; Addgene #117987) lentivirus at the highest tolerated dose, as determined by impact on growth compared to uninfected control cells.

Techniques: Western Blot, Transduction, In Situ, Recombinant, Lysis, Protease Inhibitor, Staining, Magnetic Beads, Clone Assay, Bicinchoninic Acid Protein Assay, Isolation, Labeling, Viability Assay, RNA Sequencing Assay, Sequencing, Expressing, CRISPR, Mass Spectrometry, Mutagenesis, shRNA, Plasmid Preparation, Luciferase, Software, Blocking Assay