Journal: Cell Host & Microbe

Article Title: Aspergillus fumigatus hijacks human p11 to redirect fungal-containing phagosomes to non-degradative pathway

doi: 10.1016/j.chom.2023.02.002

Figure Lengend Snippet:

Article Snippet: HCP216549-SG01-3 for generation of p11-KO cell line , GeneCopoiea , Cat# HCP216549-SG01-3.

Techniques: Virus, Recombinant, Cell Culture, Protease Inhibitor, Transfection, SYBR Green Assay, Mass Spectrometry, CyQUANT Assay, LDH Cytotoxicity Assay, cDNA Synthesis, Purification, Gene Expression, Construct, Control, Transformation Assay, Cloning, Plasmid Preparation, Expressing, Software, Imaging, Blocking Assay, Red Blood Cell Lysis, Membrane

Journal: bioRxiv

Article Title: Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

doi: 10.1101/2023.12.13.571376

Figure Lengend Snippet: Two-component vector system where Cas9 is integrated into the AAVS1 safe harbor locus to sustain stable expression. The second component is a lentiviral system containing a sgRNA targeting on and off-target sites downstream of an AcrIIA4 variant that is uniquely barcoded. B. Editing efficiency measured over time within cells expressing wild-type AcrIIA4 (circular points) versus those without wild-type AcrIIA4 (rectangular). C. Indel mutation profiles spanning the on- and off-target regions. Deletions are shown in blue and insertions are in orange. D. Experimental design in which AcrIIA4 variants were delivered to A549 cells expressing Cas9, which were cultured for 10 days prior to collection and sequencing of the AcrobaTx barcode. E. Structure of the AcrobaTx barcode and definitions of gene editing activity metrics derived from measuring editing outcomes within the on- and off-target sites. F. Editing precision of alanine scanning variants normalized to the wild-type control. D69A variant is highlighted as the variant with the highest precision within the alanine scan mutant group. G. Crystal structure of AcrIIA4 bound to the SpCas9-gRNA complex. The interaction between D69 and R1335, required for PAM recognition, is highlighted. H. Distribution of editing efficiency ( EE , left) and editing precision ( EP , right) across all AcrIIA4 variants. Variants that resulted in EP in the top 90 th percentile are highlighted in red. I. Deletion profiles of DNA repair outcomes from cells without AcrIIA4 (blue) versus cells that express a variant that promotes precise editing (red). J. Editing precision measured across AcrIIA4 variants at Day 10 versus at Day 7 (Pearson Correlation Coefficient r = 0.56).

Article Snippet: A549 cells expressing the pAG001 Cas9 vector (Genecopeia) were transduced at high MOI with 8 μg/mL polybrene using a spin-infection at 1200*g for 45 minutes.

Techniques: Plasmid Preparation, Expressing, Variant Assay, Mutagenesis, Cell Culture, Sequencing, Activity Assay, Derivative Assay, Control

Journal: bioRxiv

Article Title: Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

doi: 10.1101/2023.12.13.571376

Figure Lengend Snippet: A. Experimental design in which the top 2000 AcrIIA4 variants, rank-ordered by EP , were delivered to A549 and 293T cells for another round of screening. B. Distribution of EP across all sample groups in the second round of screening versus the first round, at left. C. Frequency of editing within the AcrobaTx barcode across seven lead candidate enAcr groups. Yellow rectangles indicate areas in which editing occurs frequently while violet depicts areas with low editing activity. D. Ratio of on- to off-target editing in cells expressing seven representative lead candidate enAcrs (purple) versus ratios observed from DNA repair outcomes within cells expressing previously described benchmark AcrIIA4 variants.

Article Snippet: A549 cells expressing the pAG001 Cas9 vector (Genecopeia) were transduced at high MOI with 8 μg/mL polybrene using a spin-infection at 1200*g for 45 minutes.

Techniques: Activity Assay, Expressing

Journal: bioRxiv

Article Title: CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

doi: 10.1101/2023.12.26.572587

Figure Lengend Snippet: (A) Correlation between log2-fold change (L2FC) in RAD51 foci number and the Rule Set 2 on-target score. All guides targeting RAD51 regulators including RAD51 paralogs (RAD51D, RAD51C, XRCC3), BRCA1, and BRCA2 in the library are shown. Splice and nonsense variants with high Rule Set 2 score shows more significant L2FC. Pearson correlation (r) equals −0.30. (B) Quantification of RAD51 foci in irradiated S/G2-phase cells with guides targeting RAD51 regulators that have low Rule Set 2 score. Cells are grouped by sgRNA category. No. or moderate significant separation from the cells with control guides is observed. Two-sided KS test, *p.adj < 0.05, **p.adj < 0.01, ***p.adj < 0.001, ****p.adj < 0.0001. (Outliers are not shown in the CDFs.) (C) as in B) for guides with high on-target score, showing significant reduction in RAD51 foci in cells with nonsense and splice guides, compared to the control guides. (D) as in A) for L2FC in BRCA1 foci for BRCA1-targeting guides, showing a strong negative correlation with the Rule Set 2 score. Pearson correlation (r) equals −0.75. (E) same as B) for BRCA1 foci and guides targeting BRCA1 that have low Rule Set 2 score, showing no significant separation from the control guides. (F) same as E) for guides with high Rule Set 2 score, showing significant reduction in BRCA1 foci in cells with missense, nonsense, and splice guides. (G) Volcano plot showing no guides targeting AAVS1 and non-targeting control (NTC) guides shows statistically significant changes in RAD51 foci under irradiation. Significance is defined by p.adj < 0.05 and absolute L2FC > 0.5. All guides targeting DDR genes with on-target score >= 0.55, all AAVS1-targeting non-targeting control (NTC) guides are shown. Benjamini-Hochberg corrected two-sided KS test. (H) Same as G) showing guides that result in significant changes in RAD51 foci. (I) Gene enrichment analysis of guides targeting RAD51D among guides causing significant changes in RAD51 foci in the irradiated condition. Fisher exact test. Enriched genes are defined as p.adj <0.05. (J) same as G) for BRCA1 foci. (K) same as H) for guides that result in significant changes in BRCA1 foci (L) same as I) for BRCA1 foci.

Article Snippet: MCF7-BE3 cells were cultured in the same medium supplemented with 2 μg/ml Blasticidin (Thermo Fisher Scientific A1113903), HT1080/Cas9 AAVS1 (Genecopoeia SL512, RRID:CVCL_C9F8) cells were cultured in the same medium supplemented with 200 μg/ml HygromycinGold B (Invivogen ant-hg-2).

Techniques: Irradiation, Control

Journal: bioRxiv

Article Title: CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

doi: 10.1101/2023.12.26.572587

Figure Lengend Snippet: (A) Wasserstein distance of cells with DDR gene-targeting guides (Perturb) or control guides (Controls) to control cells for RAD51 foci in irradiated cells. Hits of RAD51 foci identified in the pooled screening are marked. (B) same as A) for BRCA1 foci. (C) Volcano plots showing variants yielding significant changes in the proportion of cells with high p21 expression in untreated (left) or irradiated (right) conditions. Significance is defined by p.adj < 0.05, Beta-Binomial test. The proportion of control cells with high p21 is marked with the vertical line (UNT: 0.376; IR: 0.576). sgRNA category and ClinVar category for significant guides are displayed by colors and shapes, respectively. (D) Correlation of RAD51 foci log2-fold change (L2FC) in guides delivered in the pooled library (pooled) and transduced individually (individual). The Pearson correlation (r) is 0.90. (E) same as D) for BRCA1 foci. The Pearson correlation (r) is 0.95. (F) MCF7-BE3 cells were lentivirally transduced with the DDR364 library, selected in puromycin-containing medium for 2 days, and cultured 2 days without puromycin. Cells were either untreated (UNT) or treated with DNA damaging agents Camptothecin (CPT), Olaparib (OLAP), Cisplatin (CISP) or Etoposide (ETOP) for 24 hours, then fixed before optical phenotyping and barcode detection. (G) Correlation between L2FC in RAD51 foci in CISP-treated cells and the Rule Set 2 on-target score, showing a negative correlation. All guides targeting RAD51 regulators including RAD51D, RAD51C, XRCC3, BRCA1, and BRCA2 are shown. The Pearson correlation (r) equals −0.29. (H) same as G) for OLAP-treated cells. The Pearson correlation (r) equals −0.30. (I) Volcano plot showing variants yielding significant changes in RAD51 foci in CISP-treated cells. Statistical significance is defined by p.adj < 0.05 and absolute L2FC > 0.5. All guides targeting DDR genes with on-target score >= 0.5, all AAVS1-targeting and non-targeting control (NTC) guides are shown. Two-sided KS test, *p.adj < 0.05, **p.adj < 0.01, ***p.adj < 0.001, ****p.adj < 0.0001. Same figure legend as in C). (J) same as I) for OLAP-treated cells. (K) Gene enrichment analysis in variants that result in significant changes in RAD51 foci. Fisher exact test. Enrichment is defined as p.adj <0.05. (L) Same as I) for variants yielding significant changes in large γH2AX foci in CISP-treated cells. (M) same as L) for OLAP-treated cells. (N) same as K) for large γH2AX foci.

Article Snippet: MCF7-BE3 cells were cultured in the same medium supplemented with 2 μg/ml Blasticidin (Thermo Fisher Scientific A1113903), HT1080/Cas9 AAVS1 (Genecopoeia SL512, RRID:CVCL_C9F8) cells were cultured in the same medium supplemented with 200 μg/ml HygromycinGold B (Invivogen ant-hg-2).

Techniques: Control, Irradiation, Expressing, Transduction, Cell Culture

Journal: bioRxiv

Article Title: CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

doi: 10.1101/2023.12.26.572587

Figure Lengend Snippet: (A) Immunoblots on guides targeting BRCA1 showing the reduction of full-length BRCA1 protein in the splice variant BRCA1.234, compared to the missense variants BRCA1.416 and.BRCA1.476, and the AAVS1 variant AAVS1.86. Cells transduced with Firefly siRNA (siFirefly) and BRCA1 siRNA (siBRCA1) were included to show the specificity of BRCA1 detection. Cells were treated with or without irradiation and the induction of DNA damage is shown with the phospho-KAP1 (pKAP1) staining. Tubulin is used as the loading control. (B) As in A) for BRCA2 variants showing the reduction of full-length BRCA2 protein in the nonsense variant BRCA2.207, compared to the missense variant BRCA2.438 and the AAVS1 variant AAVS1.86.

Article Snippet: MCF7-BE3 cells were cultured in the same medium supplemented with 2 μg/ml Blasticidin (Thermo Fisher Scientific A1113903), HT1080/Cas9 AAVS1 (Genecopoeia SL512, RRID:CVCL_C9F8) cells were cultured in the same medium supplemented with 200 μg/ml HygromycinGold B (Invivogen ant-hg-2).

Techniques: Western Blot, Variant Assay, Transduction, Irradiation, Staining, Control

Journal: bioRxiv

Article Title: Redox-dependent formation of a viral amyloid and functional impact

doi: 10.1101/2024.01.22.576663

Figure Lengend Snippet: A) Confocal microscopy analysis of HEK293T cells transfected with either an empty vector (EV), or enhanced green fluorescent protein (EGFP), or the indicated tetracysteine ( tc )-tagged W HeV constructs. W HeV filaments were visualized 24h post-transfection with ReAsH dye to detect the tc peptide (red) and immunofluorescence staining targeting W HeV (violet). Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. B) Z-stack projections of live HEK293T cells transfected with an empty vector or the indicated tetracysteine ( tc )-tagged W HeV constructs. Cells were stained 24h and 48h post-transfection with ReAsH dye to detect the tc peptide and imaged by confocal microscopy. Images are representative of three independent experiments. Scale bar, 10 µm. C) Z-stack projections of HEK293T cells transfected with a construct driving the expression of W HeV with a C-terminal tc tag 24h post-transfection. Cells were stained and imaged as described in panel B. D) Electron micrographs of ultrathin sections at equivalent Z positions of the cells shown in C). The arrowheads indicate the fibrillar structures detected in the nuclei of the transfected cells. E) Quantification of the percentage of cells with filaments. Cells with filaments were counted from the Z-projections. Data are expressed as mean ± sd and are representative of three independent experiments with n ≥ 19 for each condition. Statistical significance was assessed with two-way ANOVA completed with a Tukey’s multiple comparison test. ****p < 0.0001.

Article Snippet: Human embryonic kidney 293T (HEK293T), A549 and the HEK293 cell line stably transduced with a firefly luciferase reporter under the control of a synthetic NF-κB promoter (HEK293-NF-κB_luc, BPS-Bioscience) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Cat# 61965-026) supplemented with 10 % fetal calf serum at 37 °C in a 5 % CO 2 incubator.

Techniques: Confocal Microscopy, Transfection, Plasmid Preparation, Construct, Immunofluorescence, Staining, Expressing, Comparison

Journal: bioRxiv

Article Title: Redox-dependent formation of a viral amyloid and functional impact

doi: 10.1101/2024.01.22.576663

Figure Lengend Snippet: A) Detection of intracellular ROS in W-transfected HEK293T cells at 24h post-transfection by confocal microscopy and B) Fluorescence intensity quantification for each condition. Data are expressed as mean ± sd (n = 9 fields of view). Statistical significance was assessed with the Kruskal-Wallis test completed by a Dunn’s multiple comparison test. Only comparisons of interest are plotted. *p<0.05, **p < 0.01. C ) Measurement of NF-κB activity in W-transfected cells. HEK293 NF-κB_luc cells were transfected with an empty vector (EV), tc -tagged W HeV constructs, or FLAG-tagged W NiV , stimulated (blue) or not (white) 24h later with 50 ng/ml IL-1β before assessing NF-κB activity by luminescence quantification. Data are expressed as mean ± sd and are representative of three independent experiments (n = 18). Statistical significance was assessed with two-way ANOVA completed with a Tukey’s multiple comparison test. Asterisks above error bars denote significance compared to the corresponding EV condition. *p<0.05, ** p < 0.01, ****p < 0.0001.Scale bar: 20 µm.

Article Snippet: Human embryonic kidney 293T (HEK293T), A549 and the HEK293 cell line stably transduced with a firefly luciferase reporter under the control of a synthetic NF-κB promoter (HEK293-NF-κB_luc, BPS-Bioscience) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Cat# 61965-026) supplemented with 10 % fetal calf serum at 37 °C in a 5 % CO 2 incubator.

Techniques: Transfection, Confocal Microscopy, Fluorescence, Comparison, Activity Assay, Plasmid Preparation, Construct