human crispr library plasmid dna  (Addgene inc)

Journal: Cell Reports Medicine

Article Title: CDK4/6 inhibition overcomes venetoclax resistance mechanisms with enhanced combination activity in acute myeloid leukemia

doi: 10.1016/j.xcrm.2025.102526

Figure Lengend Snippet: Genetic targeting of protein synthesis pathways sensitizes AML cells to venetoclax (A) Volcano plots of genome-wide CRISPR screen. All 480 genes uniquely sensitizing to ven with no evidence of an effect by combination treatment (non-significant) are shown as black dots. (B–D) Reactome pathways from differential expression analysis (DEA) of the 480 gene set show enrichment in pathways related to cell cycle (B), metabolism of RNA (C), and metabolism of proteins (D). The dot size indicates the number of evaluated genes in the Reactome pathway.

Article Snippet: Virus for sgRNA YUSA library was produced by transfecting HEK 293T/17 cells with Calcium chloride, HEPES buffer saline, VSVG, psPAX2 and human CRISPR library plasmid DNA (Addgene #67989).

Techniques: Genome Wide, CRISPR, Quantitative Proteomics

Journal: bioRxiv

Article Title: Differentiation of Toxoplasma into latent forms is linked to central carbon metabolism and requires a GID/CTLH-type E3 ubiquitin ligase

doi: 10.1101/2025.07.27.667068

Figure Lengend Snippet: (A) Schematic representation of the experimental setup. A guide library covering the full 8000 Toxoplasma genes (10 guides per gene) is transfected into tachyzoites and selected for stable integration using pyrimethamine (pyr) over three lytic cycles in HFF cells in complete media (Glc + /Gln + ). Populations were then transferred into media containing glucose (Glc + /Gln - ) or glutamine (Glc - /Gln + ) as their major carbon source and grown for a subsequent two cycles. At each stage guide pools were sampled by deep sequencing as labelled with numbers. (B) Differentially abundant hits from A (Log 2 FC (glutamine-only(sample 4) vs Glucose-only (sample 3))), graphed relative to their fitness score (Log 2 FC (Initial (sample 1) vs complete media ( sample 2) as defined by ) during standard growth conditions. Genes in red highlight those predicted to be involved in central carbon metabolism (C) Schematic of Toxoplasma central carbon metabolism with genes identified as fitness conferring in glutamine-only conditions of CRISPR screen highlighted in red. (Di) Immunofluorescence assay of WT (ME49) and ΔBFD1 parasites infecting HFF host cells and stained with GAP45 antibodies (red)(parasite periphery) and Dolichos Bifluorus Agglutinin (DBA) (Fire tone) across Alkaline stress and when carbon source was limited to glutamine and pyruvate (Glc - /Gln + /Pyr + ). (ii) Quantitation of Toxoplasma differentiation using DBA staining as a marker, comparing when parasites were exposed to complete media (Glc + /Gln + ), Alkaline stress and glutamine+ pyruvate conditions (Glc - /Gln + /Pyr + ). Mean +/− S.D plotted, n=3 biological repeats, One Way ANOVA, where **p< 0.01, ns = not significant. (iii) Quantitation of cyst wall intensity in WT and DBFD1 across conditions described in Bii. DBA intensity was calculated by measuring the mean pixel intensity of a 0.1μm thickness around the periphery of the vacuole, as marked by GAP45 and dividing this by the local mean pixel intensity of the background. Each data point represents a single parasite vacuole with the bar representing median value. Data set is a single representative experiment. Statistical testing done by ordinary one-way ANOVA where ****<0.0001, ns= not significant. (E) Quantitative Proteomic analysis of WT(ME49) and ΔBFD1 grown under conditions as marked. (i) Log-Log plot comparing changes in protein abundance when WT parasites were switched to either Alkaline Stress or Glutamine-only (Glc - /Gln - ) conditions from Complete Media (Glc - /Gln + ) for 48hrs. R 2 and slope values calculated using linear regression. (iii) Volcano plot showing changes in protein abundance of WT (ME49) parental strain versus DBFD1 under glutamine as a sole carbon source. For both ii and iii; proteins are highlighted in aqua if their encoding genes promoter binds to BFD1 (as shown by ) whilst proteins known to be tachyzoite-specific highlighted in yellow. Proteins known to be bradyzoite- and tachyzoite-specific are labelled.

Article Snippet: For each replicate, eight 60 ug aliquots of whole genome CRISPR library plasmid DNA (Addgene #80636) were linearized by AseI (NEB) digestion, ethanol precipitated, air dried and resuspended in 131 ml of cytomix (10 mM KPO4; 20 mM KCl, 5 mM MgCl 2 , 25 mM HEPES, 2 mM EGTA, pH 7.6 with KOH).

Techniques: Transfection, Sequencing, CRISPR, Immunofluorescence, Staining, Quantitation Assay, Marker, Quantitative Proteomics

Journal: bioRxiv

Article Title: Differentiation of Toxoplasma into latent forms is linked to central carbon metabolism and requires a GID/CTLH-type E3 ubiquitin ligase

doi: 10.1101/2025.07.27.667068

Figure Lengend Snippet: (A) A comparison between CRISPR fitness scores in this study versus Sidik et al .(B) Localisation of Isocitrate Dehydrogenase (IDH) (266760), Malate Dehydrogenase (MDH) (288500), Oxoglutarate/Malate Transporter (274060) and PEP carboxykinase (PEPCK)(289650) by genetic tagging with HA and mAID. MDH, OMT and PEPCK co-localised with TOM40 mitochondrial marker (Ci) GDH1 (249390) localisation by HA tagging (GDH1-HA) and subsequent knockout ΔGDH1, followed by growth in complete media (Glc + /Gln + ) or in media with glutamine as the carbon source (Glc + /Gln - ). (ii) Localisation and knockdown of PEPCK(289650) by tagging with HA and min-inducible degron (mAID) (PEPCK-mAID-HA). Addition of Indol Acetic Acid (IAA) depletes PEPCK and parasites assessed for growth in either complete media (Glc + /Gln + ) or glutamine only (Glc - /Gln + ). (iii) Quantitation of area of vacuole in mm 2 of WT(ME49) parent, GDH1-HA and ΔGDH1 in complete (Glc + /Gln + ) and glutamine-only conditions (Glc - /Gln + ). Each data point represents a single parasite vacuole with the bar representing median value. Data set is a single representative experiment to account for growth variation across experiments. Statistical testing done by ordinary one-way ANOVA where ****p<0.0005, ns= not significant. (iv) U- 13 C glutamine labelling (in the presence of pyruvate) in extracellular WT (ME49) parent vs ΔGDH1 mutant lines. The mean +/− SD plotted for n=3 biological repeats. Error bars represent multiple unpaired t-tests where *<0.05 and ns=not significant (D). Abundance of guides targeting BFD1 (200385) as measured by Log (Counts Per Million) (Log2CPM) across conditions of the CRISPR screen as shown in . Mean +/− S.D plotted, n=3 biological repeats, One Way ANOVA, where **p< 0.01, ns = not significant. (E) Principle Component Analysis (PCA) global quantitative proteomics of each biological replicate across parasite strains.

Article Snippet: For each replicate, eight 60 ug aliquots of whole genome CRISPR library plasmid DNA (Addgene #80636) were linearized by AseI (NEB) digestion, ethanol precipitated, air dried and resuspended in 131 ml of cytomix (10 mM KPO4; 20 mM KCl, 5 mM MgCl 2 , 25 mM HEPES, 2 mM EGTA, pH 7.6 with KOH).

Techniques: Comparison, CRISPR, Marker, Knock-Out, Knockdown, Quantitation Assay, Mutagenesis, Quantitative Proteomics

Journal: bioRxiv

Article Title: Differentiation of Toxoplasma into latent forms is linked to central carbon metabolism and requires a GID/CTLH-type E3 ubiquitin ligase

doi: 10.1101/2025.07.27.667068

Figure Lengend Snippet: (A) Schematic representation of sub-library CRISPR screens both in vitro and in vivo (mouse). Guide libraries targeting 66 genes (including controls) of 5 guides per gene, were constructed. For in vitro screen; the guide library was transfected into ME49:Cas9 reporter line, which expresses RFP constitutively using the Gra1 promoter ( gra1 promoter -RFP)and mNeonGreen (mNG) under control of the bradyzoite-specific BAG1 promoter ( bag1 promoter -mNG) , selected under pyrimethamine (pyr) for 3 cycles in HFF cells in standard conditions. Cultures were exposed to alkaline stress to induce differentiation for 48hrs and parasites were then FACS sorted for mNG expression and prepared for sequencing. For in vivo screen; the same sub-library was transfected into Pru:Cas9 and selected over 3 cycles on pyr for stable integration of plasmids and then injected into cohorts of Swiss mice. At day 4 post infection peritoneal exudate containing Toxoplasma was collected. At day 35 brains of mice were collected. At each step DNA was extracted and guide sequences amplified for sequencing. (B) In vitro CRISPR screen analysis; Guide frequencies at the gene level, comparing FACS sorted RFP + mNG + (Sample 3)(y-axis) and RFP + mNG - (Sample 4)(x-axis) back to the initial population (Sample 2). Each data point represents a single gene and are coloured based on predicted (or known) function, with corresponding key. (C) Generation of individual Toxoplasma mutant strains; Guide plasmids were randomly picked from sub-library and used to generate individual mutant transgenic strains in the ME49:Cas9 reporter strain . Each mutant was subjected to alkaline stress for 48hrs and FACS used to analyze differentiation into bag1 promoter -mNG expressing parasites. Samples were then normalized to parental strain. BFD1 and BFD2 were used as controls. (D) Correlation of CRISPR screen results with differentiation of individual mutants. R 2 value calculated by linear regression. (E) Results of the mouse in vivo CRISPR screen, comparing Peritoneum Fitness Score (Log 2 FC (Sample 5 vs Sample 2) (x) vs Latency Fitness Score (Log 2 FC(Sample 6 vs 5))(y) Each data point represents a single gene, colour-coded as in B. (F) Comparison of in vitro and in vivo CRISPR screens, comparing the latency fitness score (as outlined in E) vs Log 2 FC(Sample 3 vs 2) (as in B). Each data point represents a single gene colour-coded as in B.

Article Snippet: For each replicate, eight 60 ug aliquots of whole genome CRISPR library plasmid DNA (Addgene #80636) were linearized by AseI (NEB) digestion, ethanol precipitated, air dried and resuspended in 131 ml of cytomix (10 mM KPO4; 20 mM KCl, 5 mM MgCl 2 , 25 mM HEPES, 2 mM EGTA, pH 7.6 with KOH).

Techniques: CRISPR, In Vitro, In Vivo, Construct, Transfection, Control, Expressing, Sequencing, Injection, Infection, Amplification, Mutagenesis, Transgenic Assay, Comparison

Journal: bioRxiv

Article Title: Differentiation of Toxoplasma into latent forms is linked to central carbon metabolism and requires a GID/CTLH-type E3 ubiquitin ligase

doi: 10.1101/2025.07.27.667068

Figure Lengend Snippet: (A) CRISPR screen results comparing back to input library (Sample 1), showing highly fitness conferring genes that were added to the library (as determined by ). Each datapoint is an individual gene and colour coded according to the key. (B) Surveyor assay to assess gene disruption across mutants generate in as compared to parental line. (C) Waterfall plot of Peritoneum Fitness Scores (Log 2 FC(Sample 5 vs 2). n=3 biological repeats, mean+/− SD shown. (D) Waterfall plot of Latency Fitness Scores (Log 2 FC(Sample 6 vs 5). n=3 biological repeats, mean+/− SD shown.

Article Snippet: For each replicate, eight 60 ug aliquots of whole genome CRISPR library plasmid DNA (Addgene #80636) were linearized by AseI (NEB) digestion, ethanol precipitated, air dried and resuspended in 131 ml of cytomix (10 mM KPO4; 20 mM KCl, 5 mM MgCl 2 , 25 mM HEPES, 2 mM EGTA, pH 7.6 with KOH).

Techniques: CRISPR, Disruption

Journal: bioRxiv

Article Title: Differentiation of Toxoplasma into latent forms is linked to central carbon metabolism and requires a GID/CTLH-type E3 ubiquitin ligase

doi: 10.1101/2025.07.27.667068

Figure Lengend Snippet: (A) Localisation of TgGID7-HA and TgGID8-HA by immunofluorescence together with GAP45 (parasite periphery) and DAPI (Bi) Quantitative αHA Immunoprecipitation of TgGID7-HA and TgGID8-HA as compared with parental control. Statistically significant hit marked in red and other TgGID components in blue. (ii) Venn diagram comparing results across all four experiments highlighting the identification of 5 common proteins. (iii) a model of TgGID complex based on previous structural data , , . (C) Analysis of differentiation in individual CRISPR mutant lines generated in ME49:Cas9 reporter strain as determined by BAG1 promoter -mNG by FACS. Normalized to parental strain. n=3 biological replicates, One way ANOVA, where ****p<0.0001. (D) Representative images of IFA analysis of differentiation after 48hrs of alkaline stress of parent, DTgGID7 and DTgGID8 strains, where DBA(Fire tone) marks cysts wall, GAP45 marks parasite periphery and DAPI-host cell nuclei. (E) Analysis of differentiation of mutants after 48hrs of alkaline stress (i) Percentage differentiation of WT (parent) versus ΔTgGID7 and ΔBFD1 as determined by DBA cyst wall staining. n=3 biological replicates, mean +/− SD, One way ANOVA, where ****p<0.0001, ns=not significant. (ii) Percentage differentiation of WT (parent) versus ΔTgGID8 and ΔBFD1 as determined by DBA cyst wall staining. n=3 biological replicates, mean +/− SD, One way ANOVA, where ****p<0.0001. (iii) Cyst wall intensity measurements across WT, ΔBFD1, ΔTgGID7 and ΔTgGID8 as determined by mean peripheral DBA staining intensity (0.1μm outside GAP45 staining) divided by the local mean background intensity of individual parasites. Single representative experiment, where each datapoint represents a single vacuole and bar represents median value. One way ANOVA, where ****p<0.0001 and ns=not significant. (F) Analysis of differentiation of parent and ΔTgGID7 in KD3 myotubes as quantitated by DBA staining. n=3 biological replicates, mean+/− SD, Unpaired, two-tailed t-test, where ***p<0.001. (G) Log-Log plot of quantitative global proteomic analysis of WT vs ΔBFD1 vs ΔTgGID7 upon 48hrs of alkaline stress. Blue datapoint represent proteins, where their gene promoter has been shown to bind to BFD1 . Yellow datapoints represent known tachyzoite-specific proteins. Annotated proteins represent known bradyzoite and tachyzoite-specific proteins. Dotted lines represent Log 2 FC=1in both dimensions. (H) BFD1 protein levels as determined by quantitative proteomics in WT parent and ΔTgGID7 strains in both complete media (standard tachyzoite growth conditions) and alkaline stress. n=5 biological repeats, mean +/−SD. One way ANOVA, where ****p<0.0001 and ns=not significant.

Article Snippet: For each replicate, eight 60 ug aliquots of whole genome CRISPR library plasmid DNA (Addgene #80636) were linearized by AseI (NEB) digestion, ethanol precipitated, air dried and resuspended in 131 ml of cytomix (10 mM KPO4; 20 mM KCl, 5 mM MgCl 2 , 25 mM HEPES, 2 mM EGTA, pH 7.6 with KOH).

Techniques: Immunofluorescence, Immunoprecipitation, Control, CRISPR, Mutagenesis, Generated, Staining, Two Tailed Test, Quantitative Proteomics

Journal: bioRxiv

Article Title: Differentiation of Toxoplasma into latent forms is linked to central carbon metabolism and requires a GID/CTLH-type E3 ubiquitin ligase

doi: 10.1101/2025.07.27.667068

Figure Lengend Snippet: (Ai) Localisation of TgGID1-HA, TgGID8-HA and TgYPEL5-HA. (i) IFA of TgGID1-HA, TgGID7-HA, TgGID8-HA and TgGID9-HA, together with GAP45 (parasite periphery) and DAPI (B) αHA Immunoprecipitation of TgGID1-HA, TgGID8-HA and TgYPEL5-HA as compared to parental control. Statistically significant hits listed in blue and red. Blue = other GID components (C) Genotyping of individual CRISPR mutants generated in the ME49:Cas9 reporter strain , either by (i) Surveyor assay, as compared to parental control or (ii) sequencing. (iii) Gating strategy for FACS analysis of CRISPR mutants. (D) Western blot confirmation of genetic deletion of TgGID7 and TgGID8. (E) Analysis of differentiation for 48hrs in glutamine-only medium by counting DBA + vacuoles of both (i) ΔTgGID7 vs parent and (ii) ΔTgGID8 vs parent. (iii) Analysis of the mean peripheral intensity of the DBA + signal of ΔTgGID7 and ΔTgGID8 vs WT and ΔBFD1 controls as measured within a 0.1μm thickness around the boundary of the GAP45 signal. Each datapoint represents a single parasite vacuole and bar represents median data point. (iv) Quantification of vacuole size of ΔTgGID7 and ΔTgGID8 as compared to WT parent and ΔBFD1 controls. Area (μm 2 ) calculated by GAP45 staining. Graph represents a single representative experiment. One way ANOVA, where ****p<0.0001 and ns = not significant (F). Representative images of differentiation assay in KD3 myoblasts of ΔTgGID7 and WT parental controls. CST1 (fire tone), GAP45 (red), DAPI(blue). (G) Quantitative global proteomic analysis of WT, DBFD1 and ΔTgGID7 grown in complete media or under alkaline stress for 48hrs. (i) Analysis of TgGID subunit abundance in ΔTgGID7 vs WT parent samples from global proteomic analysis. Values normalized to the mean of the abundance of the TgGID subunit in WT parental samples and compared to the levels in ΔBFD1. (ii) Principal component analysis (PCA) of samples. (iii) Protein abundance of the canonical tachyzoite marker SAG1 and (iv) bradyzoite marker CST1 across all conditions. For both (ii) and (iii) n=3-5, mean +/− SD, One way ANOVA, where *p<0.05, **<0.01, ***p<0.001,****p<0.0001 and ns= not significant.

Article Snippet: For each replicate, eight 60 ug aliquots of whole genome CRISPR library plasmid DNA (Addgene #80636) were linearized by AseI (NEB) digestion, ethanol precipitated, air dried and resuspended in 131 ml of cytomix (10 mM KPO4; 20 mM KCl, 5 mM MgCl 2 , 25 mM HEPES, 2 mM EGTA, pH 7.6 with KOH).

Techniques: Immunoprecipitation, Control, CRISPR, Generated, Sequencing, Western Blot, Staining, Differentiation Assay, Quantitative Proteomics, Marker