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: bioRxiv

Article Title: Heterotypic inter-GPCR ß-arrestin coupling regulates lymphatic endothelial junctional architecture in murine lymph nodes

doi: 10.1101/435776

Figure Lengend Snippet: ( A ) Schematic of S1PR1 modulator screening system Four lentiviral vectors were transduced into U2OS cell line to enable gene activation by SAM and monitoring S1PR1 activation by TANGO system. The cells introduced with SAM sgRNA library were starved with 0.5% charcoal treated FBS, then the Venus-positive population was sorted and next-gen sequence (NGS) analysis was carried out to identify the enriched SAM sgRNA sequences. ( B ) Scatter plot showing enrichment of sgRNAs after sorting. Most sgRNAs are equally distributed in the pre-sort sample (closed gray circles) while after sorting a small fraction of sgRNAs (2,770 out of 70,290 sgRNAs) were enriched and others were not detected (open blue circles). The y-axis shows the NGS reads of sgRNAs. ( C ) Identification of top candidate genes using the MAGeCK method . The names of top ten candidate genes are indicated.

Article Snippet: The single clones were isolated from antibiotics resistant cells by limiting dilution, then introduced with the SAM sgRNA library (a gift from Feng Zhang, Addgene #1000000057) at a low multiplicity of infection.

Techniques: Activation Assay, Sequencing

Journal: Genes & Development

Article Title: Protein neddylation as a therapeutic target in pulmonary and extrapulmonary small cell carcinomas

doi: 10.1101/gad.348316.121

Figure Lengend Snippet: Whole genome CRISPR/Cas9 screen identifies potential therapeutic targets and pathways in SCLC ( A ) Schematic of the CRISPR/Cas9 dropout screen conducted using the GeCKO v2 lentiviral mouse library. ( B , C ) Volcano plots of MAGeCK analysis results from five screened mSCLC cell lines ( B ) and three screened Trp53 -null MEF isolates ( C ). ( D ) CRISPR score heat map of the top depleted and enriched genes for each of the five mSCLC cell lines and three Trp53 -null MEF isolates screened.

Article Snippet: The DNA and viral productions of both the GeCKO v2 mouse (Addgene 1000000052 and 1000000053; a gift from Feng Zhang) and human (Addgene 1000000048 and 1000000049; a gift from Feng Zhang) libraries were performed as described previously ( ).

Techniques: CRISPR, Biomarker Discovery

Journal: Genes & Development

Article Title: Protein neddylation as a therapeutic target in pulmonary and extrapulmonary small cell carcinomas

doi: 10.1101/gad.348316.121

Figure Lengend Snippet: Whole-genome CRISPR suppressor screen of MLN4924-treated MSK-LX227C cells identifies the COP9 signalosome, NF-κB, and mTOR pathways as potential mechanisms of sensitivity and resistance. ( A ) Schematic of the CRISPR/Cas9 dropout screen conducted using the GeCKO v2 lentiviral human library. ( B ) X , Y plot of average CRISPR scores for the MSK-LX227C MLN4924 (25 nM) and MSK-LX227C DMSO data sets. Enlarged markers indicate genes falling above the y = x + 1 line. Notable genes are labeled and colorized according to pathway identity ( n = 3 biological replicates for each screen data set). ( C ) Heat map of top enrichment CRISPR scores organized by select pathways for the three MSK-LX227C DMSO replicates and three MSK-LX227C MLN4924 (25 nM) replicates. ( D ) Pathway dot plot for significant enriched genes filtered by MAGeCK analysis for both the MSK-LX227C MLN4924-treated and DMSO data sets (FDR < 0.1). Pathway analysis performed with Enrichr and GO biological process gene ontology. Dot color indicates significance and dot size represents the proportion of genes identified in the pathway.

Article Snippet: The DNA and viral productions of both the GeCKO v2 mouse (Addgene 1000000052 and 1000000053; a gift from Feng Zhang) and human (Addgene 1000000048 and 1000000049; a gift from Feng Zhang) libraries were performed as described previously ( ).

Techniques: CRISPR, Labeling