crispr glycerol stock arrayed mouse sgrna library  (Merck & Co)

Journal: Nature Communications

Article Title: Genome-wide in vivo CRISPR screens identify GATOR1 complex as a tumor suppressor in Myc-driven lymphoma

doi: 10.1038/s41467-025-62615-y

Figure Lengend Snippet: A Schematic of the experimental strategy for performing in vivo genome-wide sgRNA screens to identify candidate tumor suppressors. Fetal liver cells (FLCs), a rich source of hematopoietic stem/progenitor cells (HSPCs), from E13.5 Eµ-Myc ; Cas9 embryos (C57BL/6-Ly5.2 background) were transduced with lentiviruses containing sgRNAs targeting p53 ( sgp53; positive control), a negative control sgRNA targeting human BIM ( sgControl) or a whole-genome sgRNA library 28 . The transduced FLCs were injected intravenously (i.v.) into lethally irradiated (2 × 5.5 Gy, 3 h apart) congenic recipient C57BL/6-Ly5.1 mice. Lymphoma bearing mice displayed enlarged spleen, lymph nodes and/or thymus. Genomic DNA was isolated from the spleen, comprising mostly of lymphoma cells but also containing non-transformed hematopoietic cells. The enriched sgRNAs were identified by NGS. Schematic created in BioRender. Potts, M. ( https://BioRender.com/smdpt7f ). B Tumor-free survival of mice transplanted with Eµ-Myc;Cas9 FLCs that had been lentivirally transduced with the positive control sgRNA ( sgp53) , the negative control sgRNA ( sgControl) or a whole-genome sgRNA library. The dotted line represents cut-off for lymphomas from the whole genome sgRNA library cohort that were arbitrarily deemed to be accelerated and therefore selected for further analysis. n represents total number of transplanted mice per sgRNA from 6 reconstitution cohorts. Median survival is indicated in brackets. Log-rank (Mantel-Cox) statistical test for survival curve comparison to sgControl . C Top 10 tumor suppressor genes identified as hits, determined by frequency of their sgRNAs detected in independent lymphomas from mice from the whole-genome sgRNA library cohort that showed accelerated lymphoma, with the corresponding sgRNAs found to be highly enriched ( > 50% of reads within a given lymphoma) by sequencing. Genes emboldened (five of the top 10) represent those encoding proteins with functions in the mTORC1 inhibitory pathway. Source data provided as Supplementary Data and as Source Data File.

Article Snippet: Two independent sgRNAs for in vivo validation experiments of hits and a negative control sgRNA targeting human NLRC5 , (Supplementary Table ) were obtained from the Merck CRISPR glycerol stock arrayed mouse sgRNA library available at WEHI (Sigma Aldrich # MSANGERG ).

Techniques: In Vivo, Genome Wide, Transduction, Positive Control, Negative Control, Injection, Irradiation, Isolation, Transformation Assay, Comparison, Sequencing

Journal: Nature Communications

Article Title: Genome-wide in vivo CRISPR screens identify GATOR1 complex as a tumor suppressor in Myc-driven lymphoma

doi: 10.1038/s41467-025-62615-y

Figure Lengend Snippet: A Schematic of the GATOR1 complex, consisting of three proteins, NPRL3, DEPDC5 (sgRNAs targeting their genes identified as hits in our genome-wide CRISPR screen), and NPRL2. The GATOR1 complex negatively regulates mTORC1 signaling in response to the availability of the amino acids leucine (Leu), methionine (Met) and arginine (Arg). B – D Tumor-free survival of mice transplanted with FLCs from Eµ-Myc;Cas9 E13.5 embryos that had been transduced with either the sg p53 (positive control), the negative control sgRNA ( sgControl ) or two independent sgRNAs each for targeting either Nprl3 ( B ) Depdc5 ( C ) or Nprl2 ( D ). n represents the number of transplanted mice per sgRNA across two transplanted mouse cohorts. Median survival is indicated in brackets. Two-sided log-rank (Mantel-Cox) test was used for comparison of mouse survival curves to sgControl . E , Proportions of frameshift, in frame InDels or wildtype (wt) sequence reads for the target gene of each sgRNA, analyzed by NGS. Each bar represents one lymphoma cell line derived from lymphomas of recipient mice that had been transplanted with sgNrpl3, sgDepdc5 or sgNprl2 Eµ-Myc;Cas9 FLCs ( n = 3 cell lines per genotype). Source data provided as Supplementary Data . F Survival of human patients with diffuse large B cell lymphoma (DLBCL) , a cancer driven by abnormally high c-MYC expression, stratified by GATOR1 mRNA expression, where the GATOR1 -low ( n = 103) strata is defined as expression of either the NPRL3 , DEPDC5 or NPRL2 mRNA in the lowest quartile. The others were grouped into the GATOR1 -high strata ( n = 104). Two-sided log-rank Kaplan-Meier statistical test, P = 0.0021. Source data are provided as a Source Data file.

Article Snippet: Two independent sgRNAs for in vivo validation experiments of hits and a negative control sgRNA targeting human NLRC5 , (Supplementary Table ) were obtained from the Merck CRISPR glycerol stock arrayed mouse sgRNA library available at WEHI (Sigma Aldrich # MSANGERG ).

Techniques: Genome Wide, CRISPR, Transduction, Positive Control, Negative Control, Comparison, Sequencing, Derivative Assay, Expressing