mouse crispr knockout pooled 385 library  (Addgene inc)

Journal: bioRxiv

Article Title: Genome-wide screen in Mycobacterium tuberculosis infected macrophages reveals innate regulation of antibacterial mediators by IRF2

doi: 10.1101/2025.09.26.678671

Figure Lengend Snippet: Genome-wide CRISPR knockout screen identifies regulators of TNF and iNOS in response to Mycobacterium tuberculosis infection of macrophages. (A) Schematic of screen. A genome-wide knockout library in CIMs was sorted based on TNF and iNOS expression during infection with Mtb. TNF and iNOS protein levels were analyzed by flow cytometry 24 hrs post infection and TNF + iNOS - and TNF + iNOS + CIMs were sorted as shown. (B and C) Results of genes depleted or enriched in (B) TNF + iNOS - or (C) TNF + iNOS + sorted macrophages compared to unsorted macrophages. (D and E) Gene set enrichment analysis of genes depleted in TNF + iNOS - (D) or TNF + iNOS + (E) macrophage populations. Data are the combined results of two independent experiments.

Article Snippet: The mouse Brie knockout CRISPR pooled library on the lentiGuide-Puro backbone was purchased from Addgene and amplified by the UC Berkeley High Throughput Screening Facility. sgRNAs were PCR amplified and sequenced to verify sgRNA representation on the Illumina HiSeq4000, 50SR at the UC Berkeley Genomics Sequencing Laboratory.

Techniques: Genome Wide, CRISPR, Knock-Out, Infection, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Genome-wide screen in Mycobacterium tuberculosis infected macrophages reveals innate regulation of antibacterial mediators by IRF2

doi: 10.1101/2025.09.26.678671

Figure Lengend Snippet: IRF2 inhibits proliferation during macrophage differentiation. (A) 2x10 6 double knockout CIM progenitors (transduced with guides for Irf2 or scramble and Irf9, Ifnar1 , or scramble) were differentiated for seven days and cell numbers were quantified. Data are the combined results of at least three independent experiments. Data are presented as mean +/- SD and were statistically analyzed using two-way ANOVA with Sidak’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. (B) Genome wide CRISPR knockout screen examining macrophage differentiation and proliferation. The CIM progenitor knockout library was differentiated into macrophages, and undifferentiated cells at day 0 were compared to cells differentiated for seven days. Shown are results of genes depleted or enriched in differentiated CIMs compared to progenitors. Data are the combined results of three independent experiments.

Article Snippet: The mouse Brie knockout CRISPR pooled library on the lentiGuide-Puro backbone was purchased from Addgene and amplified by the UC Berkeley High Throughput Screening Facility. sgRNAs were PCR amplified and sequenced to verify sgRNA representation on the Illumina HiSeq4000, 50SR at the UC Berkeley Genomics Sequencing Laboratory.

Techniques: Double Knockout, Transduction, Genome Wide, CRISPR, Knock-Out

Journal: bioRxiv

Article Title: Genome-wide screen in Mycobacterium tuberculosis infected macrophages reveals innate regulation of antibacterial mediators by IRF2

doi: 10.1101/2025.09.26.678671

Figure Lengend Snippet: Genome-wide CRISPR knockout screen examining macrophage differentiation and proliferation. (A) The CIM progenitor knockout library was split into two groups, group 1 was differentiated into macrophages, while group 2 was passaged in the presence of estradiol to prevent macrophage differentiation. After seven days the passaged progenitors were compared to differentiated cells. Shown are results of genes depleted or enriched in differentiate CIMs compared to progenitors. Data are the combined results of three independent experiments. (B and C) CIM progenitors lacking candidate genes were generated using two independent sgRNA for each candidate, and cells were differentiated into macrophages. After seven days all suspension and adherent cells were harvested and (B) cell numbers were quantified. (C) Collected cells were stained with antibodies for F4/80 and analyzed by flow cytometry. Data are presented as mean +/- SD and were statistically analyzed using one-way ANOVA with Sidak’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001.

Article Snippet: The mouse Brie knockout CRISPR pooled library on the lentiGuide-Puro backbone was purchased from Addgene and amplified by the UC Berkeley High Throughput Screening Facility. sgRNAs were PCR amplified and sequenced to verify sgRNA representation on the Illumina HiSeq4000, 50SR at the UC Berkeley Genomics Sequencing Laboratory.

Techniques: Genome Wide, CRISPR, Knock-Out, Generated, Suspension, Staining, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: Ints7 deficiency activates DNA damage response to elicit resurgence of endogenous retrovirus MERVL and anastasis of embryonic stem cells

doi: 10.1093/nar/gkaf797

Figure Lengend Snippet: The genome-wide screen reveals DNA damage-related pathways in the activation of MERVL . ( A ) Workflow for the genome-wide CRISPR/Cas9 screen. GeCKO, Genome-scale CRISPR-Cas9 Knockout; LTR, long terminal repeat; MOI, multiplicity of infection; FACS, fluorescence-activated cell sorting. ( B ) FACS analysis showing the percentage of MERVL -activated cells after 5 days of GeCKO library treatment. ( C ) Ranking of previously reported MERVL -negative regulators (left) and positive regulators (right). Genes are ranked by RRA score. RRA, robust ranking aggregation. ( D ) Top five Gene Ontology terms with the lowest P -value identified through the GeCKO screen. ( E ) Immunofluorescence analysis in wild-type mESCs depicting MERVL-Gag (red), γH2AX (yellow), and DAPI (gray). Bar: 10 μm. ( F ) Quantification of γH2AX fluorescence intensity in MERVL + and MERVL − cells. **** P -value < 0.0001 by t -test. ( G ) Immunofluorescence analysis demonstrating MERVL-Gag (red) in mESCs treated with DMSO and 1 and 10 μM etoposide. DMSO, dimethylsulfoxide; ETO, etoposide. Bar: 10 μm. ( H ) qPCR analysis of MERVL expression in mESCs treated with DMSO and 1 and 10 μM etoposide. * P -value < 0.05, **** P -value < 0.0001 by one-way ANOVA, error bars represent the SD. ( I ) Western blot analysis of MERVL-Gag and γH2AX in mESCs collected 1 day after 7 Gy of X-ray irradiation or untreated controls.

Article Snippet: The plasmid DNA library was amplified following the protocol provided by Addgene ( http://www.addgene.org/pooled-library/broadgpp-mouse-knockout-brie/ ).

Techniques: Genome Wide, Activation Assay, CRISPR, Knock-Out, Infection, Fluorescence, FACS, Immunofluorescence, Expressing, Western Blot, Irradiation