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

Journal: Cell reports

Article Title: Identification of modulators of the ALT pathway through a native FISH-based optical screen

doi: 10.1016/j.celrep.2024.115114

Figure Lengend Snippet: (A) Rolling circle assay (RCA) analysis of genomic DNA isolated from U2OS cells transfected with the sgRNA and relative quantification. (B) Example of TAILS data and quantification; U2OS cells transfected with indicated sgRNA were processed as described in the TAILS pipeline. A minimum of 2,200 cells were analyzed per condition. The scale bar represents 10 μm. (C) TAILS analysis in ALT-positive U2OS cells. The ssTelo intensity derived from two biological replicates of the arrayed sgRNA library. Linear regression analysis by Pearson correlation coefficient (R) was calculated, and it is 0.701. (D) Scatterplot displaying mean Z score for each gene assayed (y axis) and the relative ranking based on descending Z score (x axis). Dotted lines indicate the cutoff value (±2.4) chosen to identify putative hits. Genes that have previously been reported to affect ALT activity are indicated. (E) Images obtained by TAILS of hits selected known modulators of ALT. The scale bar represents 10 μm. (F) Gene function classification of all the genes contained in sgRNA library (left diagram) and the hits identified by TAILS (right diagrams). For more information, see .

Article Snippet: The arrayed sgRNA library (Synthego) used in this study targets 1064 human genes involved in DNA transactions (see for details).

Techniques: Isolation, Transfection, Quantitative Proteomics, Derivative Assay, Activity Assay

Journal: Cell reports

Article Title: Identification of modulators of the ALT pathway through a native FISH-based optical screen

doi: 10.1016/j.celrep.2024.115114

Figure Lengend Snippet: (A) Gene function composition of the arrayed sgRNA library (“Library”) and the number genes that were identified as putative ALT suppressors by TAILS (“Enriched”). (B) List of ALT-suppressor genes that were further characterized in this study. The table reports the average Z score (“Score”) and gene function category (“Function”). (C and D) Rolling circle assay (RCA) analysis of genomic DNA isolated from 3 independent DDX39A −/− clones (C1, C2, and C3) and the parental U2OS cells (WT). (E and F) Representative images and quantification of DDX39A-deficient and -proficient cells in ALT-positive U2OS and ALT-negative HeLa backgrounds. Each dot represents one cell, with a minimum of 200 cells per condition analyzed across 2 independent experiments. The scale bar represents 10 μm. (G) ssTelo staining of U2OS cells transfected with sgRNAs against FANCM, DDX39A, DDX39B, and a non-targeting control (sgCtrl) in the presence of absence of sgRNA against BLM. A minimum of 1,800 cells were analyzed per condition. The scale bar represents 10 μm. (H) Quantification of the data shown in (G). (I and J) Representative images and quantification of ssTelo analysis ALT-positive U2OS, G292, and SAOS-2 cells treated with transcription inhibitor DRB. Each dot represents one cell, with a minimum of 250 cells per condition analyzed across 2 independent experiments. The scale bar represents 10 μm. (K) Quantification of ssTelo analysis U2OS cells treated with DRB in the presence of absence of sgRNA against BLM. Each dot represents one cell, with a minimum of 300 cells per condition analyzed across 2 independent experiments. For representative images, see . Data are represented as mean ± SEM. An unpaired t test was used for statistical analysis; ** p ≤ 0.01 and **** p ≤ 0.0001 on the graphs.

Article Snippet: The arrayed sgRNA library (Synthego) used in this study targets 1064 human genes involved in DNA transactions (see for details).

Techniques: Isolation, Clone Assay, Staining, Transfection, Control

Journal: Cell reports

Article Title: Identification of modulators of the ALT pathway through a native FISH-based optical screen

doi: 10.1016/j.celrep.2024.115114

Figure Lengend Snippet:

Article Snippet: The arrayed sgRNA library (Synthego) used in this study targets 1064 human genes involved in DNA transactions (see for details).

Techniques: Recombinant, Extraction, SYBR Green Assay, Control, Blocking Assay, Reverse Transcription, shRNA, Plasmid Preparation, Software

Journal: Plant Methods

Article Title: An efficient multiplex approach to CRISPR/Cas9 gene editing in citrus

doi: 10.1186/s13007-024-01274-4

Figure Lengend Snippet: RPS5a driving zCas9i increases gene editing efficiency in a multiplex system in Carrizo citrange. A Diagram of the vectors containing an ES8Z promoter driving the tRNA cassette with the four sgRNAs and number of plants genotyped for each vector. B Heatmaps of the knockout scores for the targets of each sgRNA in individual plants. Indicated by a colored box. Grey boxes = no data, X = failed sequencing. Knockout scores are the percentage of sequence likely to disrupt a CDS reading frame estimated by the ICE Analysis tool (v3.0) (Synthego). C Proportion of individual plants in a population being classified into quartiles by knockout score, for each of the four sgRNA. D Average knock-out scores (ICE Synthego score) of Sanger sequencing genotyping showing variation among sequenced samples (78, 58, and 88 plants) for each position of sgRNA for the lines shown in panel ( B ). E Editing scores of the pooled whole genome sequencing of each sgRNA for all tested vectors

Article Snippet: This was done for the RUBY gene, amplified from the 35S :: Ruby plasmid (Addgene plasmid #160908) [ ] using primers getRuby_F and getRuby_R, as well as synthesized arrays of sgRNAs separated by tRNA sequences ( Arabidopsis thaliana , Glycine, GCC anticodon) (Gene Universal Inc. Newark, DE, USA).

Techniques: Multiplex Assay, Plasmid Preparation, Knock-Out, Sequencing