Journal: Cell Discovery

Article Title: Spatially resolved C1QC + macrophage-CD4 + T cell niche in colorectal cancer microenvironment: implications for immunotherapy response

doi: 10.1038/s41421-025-00811-2

Figure Lengend Snippet: a Schematic representation of the workflow for the genome-wide CRISPR/Cas9 screen. b Cumulative distribution function (CDF) of biological replicates of 5 representative samples of M0, MHC low , and MHC high cells. CPM, counts per million. n = 5 in each group. c Top hit genes of MHC low and MHC high cells. The P value was corrected by Benjamini-Hochberg test. d Gene ontology (GO) term analysis of MHC low and MHC high cells. e , f KEGG gene interaction network of the hit genes in MHC low and MHC high cells. Subnetworks (Neighborhoods) are colored and annotated with enriched functional categories. Gray lines, connections within a neighborhood; red lines, connections between neighborhoods. g , h Representative flow cytometric plot and quantification of MHC-II expression levels in ESRRA -KO THP-1 cells during macrophage polarization. Vector control and THP-1-Cas9 were used as control for comparison. Vector control indicates THP-1 cells infected with lentivirus carrying an empty plasmid lacking gRNA. gMFI, geometric mean fluorescence intensity. P values in h were determined using one-way ANOVA.

Article Snippet: Human CRISPR Brunello lentiviral pooled library (Addgene, Plasmid #73138) consisting of 76,441 gRNAs was co-transfected with packaging plasmids (psPAX2 and pMD2.G) into HEK293T cells using LipoD 293T transfection reagent (Signagen, #SL100668) following the manufacturer’s protocol.

Techniques: Genome Wide, CRISPR, Functional Assay, Expressing, Plasmid Preparation, Control, Comparison, Infection, Fluorescence

Journal: Nature Communications

Article Title: Integrative oncogene-dependency mapping identifies RIT1 vulnerabilities and synergies in lung cancer

doi: 10.1038/s41467-021-24841-y

Figure Lengend Snippet: a 96-hour dose–response of isogenic PC9 cells to erlotinib alone (left panel) or to a 1:1 molar ratio of erlotinib and torin1 (right panel). Fraction of viable cells was determined using CellTiterGlo and normalized to the average value in DMSO treated cells. Data shown are the mean ± s.d. of 8 technical replicates. b Schematic of the genome-wide CRISPR-Cas9 screens performed in isogenic PC9 cells. Created with BioRender.com. c , Proliferation rates of the PC9-Cas9 isogenic cells used for genome-wide screening in 50 nM erlotinib. Data shown are the mean ± 95% confidence interval for two replicates per cell line. d Box plot showing sgRNA abundance (log 2 reads per million) of sgRNAs targeting each indicated gene. Abundance in the plasmid library, early time point (ETP), or after 12 population doublings in vehicle (DMSO) or erlotinib was determined by PCR and Illumina sequencing. Box plots show the median (center line), first and third quartiles (box edges), and the min and max range (whiskers) of replicates. * p = 0.022 (sgKRAS), ** p = 0.009 (sgRIT1), * p = 0.004 (sgEGFR) calculated by one-sided permutation testing using MAGeCK. For control PC9 cells ETP and DMSO, n = 3 biological replicates. For oncogene-expressing PC9 cells DMSO and erlotinib, n = 2 biological replicates. Source data are provided as a Source Data file.

Article Snippet: The human CRISPR Brunello lentiviral pool was obtained from the Broad Institute Genetic Perturbation Platform and is also available from Addgene (73179-LV).

Techniques: Genome Wide, CRISPR, Plasmid Preparation, Illumina Sequencing, Control, Expressing

Journal: Nature Communications

Article Title: Integrative oncogene-dependency mapping identifies RIT1 vulnerabilities and synergies in lung cancer

doi: 10.1038/s41467-021-24841-y

Figure Lengend Snippet: a Venn Diagram showing the number of significant essential genes (CRISPR Score < 0.5 and p < 0.05) shared or specific to each isogenic PC9 cell line in erlotinib. b MSigDB overlap analysis of the enriched KEGG gene sets in the shared 813 genes from ( a ). c – e , Rank plots of CRISPR scores (CS) of erlotinib-treated vs. starting plasmid in ( c ), PC9-Cas9-EGFR T790M/L858R ( d ), PC9-Cas9-KRAS G12V , and ( e ), PC9-Cas9-RIT1 M90I . Key dependencies discussed in the text are labeled. Gray dashed lines mark genes with |CS| > 0.5. f – n Box plot showing the sgRNA abundance (log 2 reads per million) of sgRNAs targeting each indicated gene. Abundance in the plasmid library, early time point (ETP), or after 12 population doublings in vehicle (DMSO) or erlotinib was determined by PCR and Illumina sequencing. Box plots show the median (center line), first and third quartiles (box edges), and the min and max range (whiskers) of replicates. f – h sgRNAs targeting PIK3CA (* p = 0.022), ILK (** p = 0.006 ) , or LIMS1 (** p = 0.005) in control or PC9-Cas9-EGFR T790M/L858R cells. i – k sgRNAs targeting ICMT (* p = 0.025), CAMK2G (* p = 0.045), or PDE11A (** p = 0.004) in control or PC9-Cas9-KRAS G12V . l – n sgRNAs targeting IGF1R (* p = 0.023), FURIN (** p = 0.003), or CPD (** p = 0.009) in control or PC9-Cas9-RIT1 M90I cells. * p < 0.05, ** p < 0.01, calculated by one-sided permutation testing using MAGeCK. For control PC9 cells ETP and DMSO, n = 3 biological replicates. For oncogene-expressing PC9 cells DMSO and erlotinib, n = 2 biological replicates. Source data are provided as a Source Data file.

Article Snippet: The human CRISPR Brunello lentiviral pool was obtained from the Broad Institute Genetic Perturbation Platform and is also available from Addgene (73179-LV).

Techniques: CRISPR, Plasmid Preparation, Labeling, Illumina Sequencing, Control, Expressing

Journal: Nature Communications

Article Title: Integrative oncogene-dependency mapping identifies RIT1 vulnerabilities and synergies in lung cancer

doi: 10.1038/s41467-021-24841-y

Figure Lengend Snippet: a – c Volcano plots of genome-wide CRISPR screening data from ( a ) PC9-Cas9-EGFR T790M/L858R cells, ( b ) PC9-Cas9-KRAS G12V cells and ( c ) PC9-Cas9-RIT1 M90I cells cultured in 40 nM erlotinib for 12 population doublings. CRISPR Score indicates the normalized log 2 (fold-change) of the average of 4 sgRNAs per gene in two biological replicates in erlotinib compared to the starting abundance in the plasmid library. d MSigDB overlap analysis of the top REACTOME gene sets in RIT1 M90I positively selected gene knockouts ( p < 0.05, CRISPR score > 0.5). e Depiction of positively selected (green) and negatively selected (blue) Hippo pathway components identified in PC9-Cas9-RIT1 M90I (|CS| > 0.5 and p < 0.05). Schematic created partially with Biorender.com. f Phase contrast images of SALEiCas9 cells (control) or expressing RIT1 M90I , the constitutively nuclear-localized YAP1 5SA , or RIT1 M90I and YAP1 5SA . Scale bar = 200 µm. Representative images from n = 3 independent experiments are shown. g Xenograft assay of SALEiCas9 cells expressing RIT1 M90I , YAP1 5SA , or combined RIT1 M90I and YAP1 5SA in immunocompromised mice. Data shown are the mean ± s.e.m. of n = 6 tumors per group. * p < 0.05 by unpaired two-tailed t-test. h Xenograft assay of SALEiCas9 cells expressing sgNF2, or combined RIT1 M90I and sgNF2 in immunocompromised mice. Data shown are the mean ± s.e.m. of n = 8 tumors per group. * p < 0.05 by unpaired two-tailed t-test. i - l Xenograft assays of SALEiCas9-RIT1 M90I + YAP1 5SA cells transduced with non-targeting control sgRNAs (sgNTC) or RIT1 -, AURKA -, IGF1R , or FURIN -targeting sgRNAs ( n = 8 tumors per sgRNA). Cells were pre-treated with doxcycyline in vitro for 5 days and re-induced at day 26 (arrow) in vivo with doxycycline-containing chow. * p < 0.05 by unpaired two-tailed t-test. Source data are provided as a Source Data file.

Article Snippet: The human CRISPR Brunello lentiviral pool was obtained from the Broad Institute Genetic Perturbation Platform and is also available from Addgene (73179-LV).

Techniques: Genome Wide, CRISPR, Cell Culture, Plasmid Preparation, Control, Expressing, Xenograft Assay, Two Tailed Test, Transduction, In Vitro, In Vivo

Journal: Molecular cancer research : MCR

Article Title: Inactivation of NF1 promotes resistance to EGFR inhibition in KRAS/NRAS/BRAF V600 -wildtype colorectal cancer

doi: 10.1158/1541-7786.MCR-19-1201

Figure Lengend Snippet: A. Schematic outline of the CRISPR modifier screen. DIFI colorectal cancer cells were transduced with lentiviral particles encoding for the expression of the Cas9 gene. DIFI-Cas9 cells were then transduced with lentiviral particles for the Brunello library of 77,440 sgRNAs targeting 19,110 genes. Cells were cultured in the presence of either DMSO or 240 nM gefitinib for up to 8 population doublings, genomic DNA was then purified and sgRNAs amplified by PCR and sequenced. sgRNAs were identified, mapped to their target genes and reads quantified.

Article Snippet: Lentiviral production To generate the lentiviral particles for the Brunello whole genome library (Addgene #73178) containing 77,440 sgRNAs targeting 19,110 genes, ( 12 , 13 ), HEK293T cells were seeded at 40 % confluence in T225 flasks.

Techniques: CRISPR, Transduction, Expressing, Cell Culture, Purification, Amplification