mouse crispr gecko v2 pooled library  (Addgene inc)

Journal: Genome Biology

Article Title: In vivo perturb-seq of cancer and microenvironment cells dissects oncologic drivers and radiotherapy responses in glioblastoma

doi: 10.1186/s13059-024-03404-6

Figure Lengend Snippet: CRISPRi screens identify oncogenic drivers and modifiers of radiotherapy response in GBM. A Schematic of in vitro screen workflow to nominate candidates for perturb-seq. B Kaplan–Meier survival curves of GL261 intracranial tumor models treated with either fractionated radiotherapy (2 Gy × 5 fractions) to the whole brain or no treatment. p value derived from log-rank test. C Volcano plot of log2 enrichment for the top 3 sgRNAs corresponding to each gene target in the growth (left) or radiation to growth ratio (right) phenotypes in GL261 cells. A discriminant threshold score of 7 was used for both screen analyses to nominate hit genes for in vivo perturb-seq. MW = Mann-Whitney U . D Gene ontology enrichment analysis for negative growth (left) and negative radiation to growth ratio (right) screen hits identified in C

Article Snippet: Protospacer sequences were selected from the optimized mouse CRISPRi v2 library [ ], and the top two scoring sgRNAs for each target gene based on depletion or enrichment phenotypes were cloned into dual sgRNA lentivirus expression vectors with direct capture tags (Addgene 187241) [ ] using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs #E2621L) (Table S2).

Techniques: In Vitro, Derivative Assay, In Vivo, MANN-WHITNEY

Journal: Genome Biology

Article Title: In vivo perturb-seq of cancer and microenvironment cells dissects oncologic drivers and radiotherapy responses in glioblastoma

doi: 10.1186/s13059-024-03404-6

Figure Lengend Snippet: In vitro perturb-seq against oncogenic drivers and modifiers of radiotherapy response in GBM cells. A Schematic of in vitro perturb-seq workflow against target genes nominated by genome-scale CRISPRi screens, combined with treatment with or without 2 Gy × 5 fractions of radiotherapy. B Distribution of mean on-target knockdown levels for all in vitro perturbations in either treatment condition. Box plots show 1st quartile, median, and 3rd quartile; whiskers represent 1.5 inter-quartile range. p values indicate Mann–Whitney U test compared to non-targeting controls. C Bubble plot of gene set enrichment analyses of gene expression modules (rows) following sgRNA perturbations (columns) without radiotherapy. Expression represents log 2 fold change normalized to non-targeting controls. Top bar charts show mRNA remaining of target gene, growth (gamma), radiation (tau), and radiation to growth ratio (rho) in vitro screen phenotypes. Gene ontology of perturbed target genes indicated. D As in C but in radiotherapy conditions. Perturbation phenotypes were normalized to irradiated cells expressing non-targeting sgRNAs. E LDA UMAP plots showing distribution of single cells expressing the indicated sgRNAs in either no radiotherapy (top) or radiotherapy (bottom) conditions. Cells which express sgRNAs other than the one highlighted in color are indicated in gray. F Difference in UMAP gaussian kernel densities between perturbations in radiotherapy (RT) and no treatment (no RT) conditions. Each point represents a particular genetic perturbation. Gray bar = mean

Article Snippet: Protospacer sequences were selected from the optimized mouse CRISPRi v2 library [ ], and the top two scoring sgRNAs for each target gene based on depletion or enrichment phenotypes were cloned into dual sgRNA lentivirus expression vectors with direct capture tags (Addgene 187241) [ ] using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs #E2621L) (Table S2).

Techniques: In Vitro, Knockdown, MANN-WHITNEY, Gene Expression, Expressing, Irradiation

Journal: Genome Biology

Article Title: In vivo perturb-seq of cancer and microenvironment cells dissects oncologic drivers and radiotherapy responses in glioblastoma

doi: 10.1186/s13059-024-03404-6

Figure Lengend Snippet: In vivo perturb-seq against oncogenic drivers and modifiers of radiotherapy response using pre-infected GL261 cells. A Schematic of in vivo pre-infected perturb-seq workflow against target genes nominated by large scale CRISPRi screens, combined with treatment with or without 2 Gy × 5 fractions of radiotherapy. B Integrated scRNA-seq UMAP of malignant and stromal/microenvironment cells from orthotopic tumors containing pre-infected GL261 cells, including sorted and unsorted cells. C Integrated scRNA-seq UMAP from B overlayed with BFP (marker for dCas9-KRAB; left) or sgRNA (right) expression levels. D Distribution of mean on-target knockdown levels for all in vivo pre-infected perturbations in either treatment condition. Box plots show 1st quartile, median, and 3rd quartile; whiskers represent 1.5 inter-quartile range. p values indicate Mann–Whitney U test compared to non-targeting controls. E Bubble plot of gene set enrichment analyses of gene expression modules (rows) following sgRNA perturbations (columns) without radiotherapy for in vivo pre-infected perturb-seq. Expression represents log 2 fold change normalized to non-targeting controls. Top bar charts show mRNA remaining of target gene, growth (gamma), radiation (tau), and radiation to growth ratio (rho) in vitro screen phenotypes. Gene ontology of perturbed target genes indicated. F As in E but in radiotherapy conditions. Perturbation phenotypes were normalized to cells from irradiated tumors that expressed non-targeting sgRNAs. G LDA UMAP plots showing distribution of single cells from in vivo pre-infected experiments expressing the indicated sgRNAs in either no radiotherapy (top) or radiotherapy (bottom) conditions. Cells which express sgRNAs other than the one highlighted in color are indicated in gray. H Difference in UMAP gaussian kernel densities between perturbations in radiotherapy (RT) and no treatment (no RT) conditions. Each point represents a particular genetic perturbation. Gray bar = mean

Article Snippet: Protospacer sequences were selected from the optimized mouse CRISPRi v2 library [ ], and the top two scoring sgRNAs for each target gene based on depletion or enrichment phenotypes were cloned into dual sgRNA lentivirus expression vectors with direct capture tags (Addgene 187241) [ ] using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs #E2621L) (Table S2).

Techniques: In Vivo, Infection, Marker, Expressing, Knockdown, MANN-WHITNEY, Gene Expression, In Vitro, Irradiation

Journal: Genome Biology

Article Title: In vivo perturb-seq of cancer and microenvironment cells dissects oncologic drivers and radiotherapy responses in glioblastoma

doi: 10.1186/s13059-024-03404-6

Figure Lengend Snippet: In vivo perturb-seq after tumor establishment using convection enhanced delivery. A Schematic of in vivo CED perturb-seq workflow against target genes nominated by genome-scale CRISPRi screens, combined with treatment with or without 2 Gy × 5 fractions of radiotherapy. B Integrated scRNA-seq UMAP of malignant and stromal/microenvironment cells from orthotopic GL261 tumors following CED, including sorted and unsorted cells. C Confocal image of GL261 orthotopic tumor transduced with a sgRNA library tagged with EGFP. Scale bar, 100 μm. D Integrated scRNA-seq UMAP of malignant and stromal/microenvironment cells from B overlayed with BFP (marker for dCas9-KRAB; left) or sgRNA (right) expression levels. E Distribution of mean on-target knockdown levels for all in vivo CED perturbations in either treatment condition. Box plots show 1st quartile, median, and 3rd quartile; whiskers represent 1.5 inter-quartile range. p values indicate Mann–Whitney U test compared to non-targeting controls. F Bubble plot of gene set enrichment analyses of gene expression modules (rows) following sgRNA perturbations (columns) without radiotherapy for in vivo CED perturb-seq. Expression represents log 2 fold change normalized to non-targeting controls. Top bar charts show mRNA remaining of target gene, growth (gamma), radiation (tau), and radiation to growth ratio (rho) in vitro screen phenotypes. Gene ontology of perturbed target genes indicated. G As in E but in radiotherapy conditions. Perturbation phenotypes were normalized to cells from irradiated tumors that expressed non-targeting sgRNAs following CED. H L2-norm synergy scores for each sgRNA perturbation across all experimental contexts (in vitro, in vivo pre-infected, in vivo CED)

Article Snippet: Protospacer sequences were selected from the optimized mouse CRISPRi v2 library [ ], and the top two scoring sgRNAs for each target gene based on depletion or enrichment phenotypes were cloned into dual sgRNA lentivirus expression vectors with direct capture tags (Addgene 187241) [ ] using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs #E2621L) (Table S2).

Techniques: In Vivo, Convection, Transduction, Marker, Expressing, Knockdown, MANN-WHITNEY, Gene Expression, In Vitro, Irradiation, Infection

Journal: STAR Protocols

Article Title: Protocol for in vivo CRISPR screening targeting murine testicular cells

doi: 10.1016/j.xpro.2024.103306

Figure Lengend Snippet:

Article Snippet: GeCKO v2 mouse CRISPR knockout pooled library , Sanjana et al. , Addgene: #1000000052 and #1000000053.

Techniques: Recombinant, Cell Counting, Whole Genome Amplification, CRISPR, Knock-Out, Software