human gecko lentiviral sgrna library v2 lenticrispr  (Addgene inc)

Journal: mBio

Article Title: CRNKL1 Is a Highly Selective Regulator of Intron-Retaining HIV-1 and Cellular mRNAs

doi: 10.1128/mBio.02525-20

Figure Lengend Snippet: Genome-wide CRISPR/Cas screen. (A) Outline of screening strategy. (B) Flow cytometric analysis of J-dual#3 and J-dual#6 reporter cells without transduction and 4 days after transduction by the GeCKO v2 lentiviral vector library (MOI, around 2) immediately prior to the sorting process. The total number of events shown in the graph, total number of cells analyzed in the screen, and total numbers of cells sorted are indicated. (C) Enrichment analysis for guide RNAs in transduced and selected J-dual#3 and J-dual#6 reporter cells compared to transduced but unselected Jurkat cells by the MAGeCK program. The robust ranking aggregation (RRA) score reflects the enrichment of guide RNA targeting the same gene in cells selected from both reporter cell clones. The distribution of the negative log 10 of the RRA score for all targeted genes in alphabetical order on the x axis is shown, with the top 12 candidate genes highlighted. (D) Additional MAGeCK ranking parameters of the top 12 candidate genes. fdr, false discovery rate; # sgRNA, number of enriched guide RNAs targeting the same gene.

Article Snippet: Individual CRISPR knockout plasmids were constructed based on lentiCRISPRv2 (no. 52961; Addgene) and GeCKO v2 sgRNA target sequences.

Techniques: Genome Wide, CRISPR, Transduction, Plasmid Preparation, Clone Assay

Journal: eLife

Article Title: TMEM79/MATTRIN defines a pathway for Frizzled regulation and is required for Xenopus embryogenesis

doi: 10.7554/eLife.56793

Figure Lengend Snippet: ( A ) An outline of the screening strategy for negative components of Wnt/β-catenin signaling using a HEK293T-TOP-EGFP reporter line. ( B ) Representative images of the reporter cells with or without infection of the GeCKO library. GFP + cells were enriched by FACS. Scale bar, 200 µm. ( C ) Scatter plots showing candidate genes corresponding to sgRNAs enriched. Results of two independent screens are shown. Each dot represents a candidate gene. Y-axis: number of unique sgRNAs for each gene (six sgRNAs per gene in the GeCKO library). X-axis: sgRNA NGS (next generation sequencing) reads for each gene. ( D ) Schematic diagram of TMEM79 protein (predicted). Y280*, a nonsense mutation at tyrosine 280 coded by the matted allele in mice. Figure 1—source data 1. Raw data for .

Article Snippet: We performed positive selection screen for negative Wnt/β-catenin regulators following published protocols using human GeCKO v.2 sgRNA library (Addgene #1000000049) ( ).

Techniques: Infection, Next-Generation Sequencing, Mutagenesis