Journal: Nature

Article Title: Saturation Editing of Genomic Regions by Multiplex Homology-Directed Repair

doi: 10.1038/nature13695

Figure Lengend Snippet: (a) Experimental schematic. Cultured cells were co-transfected with a single Cas9-sgRNA construct (CRISPR) and a complex homology-directed repair (HDR) library containing an edited exon that harbors a random hexamer (blue, green, orange) and a fixed selective PCR site (red). CRISPR-induced cutting stimulated homologous recombination with the HDR library, inserting mutant exons into the genomes of many cells. At five days post-transfection, cells were harvested for gDNA and RNA. After reverse transcription, selective PCR was performed followed by sequencing of gDNA and cDNA derived amplicons. Hexamer enrichment scores were calculated by dividing cDNA counts normalized by gDNA counts. (b) Correlation of enrichment scores between biological replicates for hexamers observed in each experiment with positions of previously identified exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs) and stop codons indicated. (c) Rank-ordered plot of enrichment scores with positions of ESEs, ESSs, and stop codons indicated.

Article Snippet: A bicistronic Cas9-sgRNA vector designed to cleave within BRCA1 exon 18 (“pCas9-sgBRCA1x18”) was cloned according to a published protocol by ligating annealed oligonucleotides into a human codon-optimized S. pyogenous Cas9-sgRNA vector from the lab of Feng Zhang (pX330-U6-Chimeric_BB-CBh-hSpCas9; Addgene plasmid #42230).

Techniques: Cell Culture, Transfection, Construct, CRISPR, Random Hexamer Labeling, Homologous Recombination, Mutagenesis, Sequencing, Derivative Assay

Journal: bioRxiv

Article Title: CRISPR activation screens map the genomic landscape of cancer glycome remodeling

doi: 10.1101/2025.05.26.656133

Figure Lengend Snippet: A) Inhibition of anticancer immunity by Siglec receptors. Siglecs bind to sialic acid-containing glycans on the surface of target cells. B) Components of the Siglec-7 ligand structure. Disialyl-T glycans are organized in dense arrays on the backbone of specific mucin-type O-glycoproteins. C ) Targeted overexpression of genes by CRISPRa. A cell line expressing a dCas9-GCN4 peptide array and a VP64-SunTag fusion protein serves as the target cell line (K-562-CRISPRa cells). This cell line is lentivirally transduced with an sgRNA targeting the promoter region of a target gene. Subsequent recruitment of chromatin remodeling factors induces an increase in transcriptional activity. D) Recombinant Siglec-Fc proteins were precomplexed with an AlexaFluor647-antihuFc antibody (1 μg/mL) for 1 hour on ice. Precomplexes were subsequently incubated with K-562 cells for 30 minutes and analyzed by flow cytometry. Representative flow cytometry plots are depicted for each Siglec-Fc as well as a human Fc (hFc) negative control. E) K-562-CRISPRa cells were lentivirally transduced with a genome-wide library of ∼104,000 sgRNAs (5 sgRNAs/gene). After selection and propagation, 1.25 x 10 8 cells were then stained with Siglec-Fc reagents as in B . A high-binding (top 20%) and low-binding (bottom 20%) of the fluorescent population was then selected and sorted by FACS.

Article Snippet: The CRISPRa-v2 library (top 5 sgRNAs/gene) containing 104,535 sgRNAs was purchased from Addgene (Pooled Library 83978).

Techniques: Inhibition, Over Expression, Expressing, Peptide Microarray, Transduction, Activity Assay, Recombinant, Incubation, Flow Cytometry, Negative Control, Genome Wide, Selection, Staining, Binding Assay

Journal: bioRxiv

Article Title: CRISPR activation screens map the genomic landscape of cancer glycome remodeling

doi: 10.1101/2025.05.26.656133

Figure Lengend Snippet: A) K-562-dCas9-CRISPRa cells were transduced with two different sgRNAs targeting the CD24 promoter region. After selection with puromycin, cells were stained with Siglec-10-Fc as in 1B and with a fluorescent antibody against CD24. Representative flow cytometry plots for both stains are shown. B) THP-1 cells were stained with Siglec-10-Fc as in 1B and a fluorescent antibody against CD24. Representative flow cytometry plots for both stains are shown. C) OCI-AML-2-Cas9 cells were lentivirally transduced with an sgRNA targeting MGAT1 to generate a polyclonal cell population containing WT and MGAT1 KO cells. Cells were then co-stained with Siglec-10-Fc and the lectin L-PHA (5 μg/mL), which binds to complex N-linked glycans. Representative staining is indicated on the flow cytometry plot. D) Graph indicates MFI of Siglec-10-Fc staining in the WT (L-PHA+) and MGAT1 KO (L-PHA-) populations. E) OCI-AML-2-Cas9 cells were lentivirally transduced with an sgRNA targeting C1GALT1. Cells were co-stained with Siglec-10-Fc and the lectin DBA, which binds to exposed α-GalNAc. Representative staining is indicated on the flow cytometry plot. F) Graph indicates MFI of Siglec-10-Fc staining in the WT (DBA-) and C1GALT1 KO (DBA+) populations. G) Pathway diagram indicates the enzymes involved in 2,3-linked and 2,6-linked sialylation of N-linked glycans. ST3GAL4, ST3GAL6 and ST6GAL1 were all strong hits in the Siglec-10 screen. H) The heat map indicates the average mRNA expression of key glycosyltransferase hits in cell lines derived from B-ALL, T-ALL, AML and multiple myeloma. mRNA expression values were extracted from DepMap and the Human Protein Atlas. I) OCI-AML-2-Cas9 cells were transduced an sgRNAs against ST3GAL4. Cells were then stained with fluorescently labeled Siglec-10-Fc. A representative flow cytometry plot is shown. J) The MFI of Siglec-10 staining in OCI-AML-2 WT and ST3GAL4 KO cells is indicated. MFI is internally normalized to the WT cell line. K) MM1S-Cas9 cells were transduced an sgRNAs against ST6GAL1. ST6GAL1 KO cells were isolated by FACS using the lectin SNA. Cells were then stained with fluorescently labeled Siglec-10-Fc. A representative flow cytometry plot is shown. L) The MFI of Siglec-10 staining in MM1S WT and ST6GAL1 KO cells is indicated. MFI is internally normalized to the WT cell line. M) K-562-CRISPRa cells were transduced and stained as in 4C . A representative flow cytometry plot is shown comparing Siglec-10-Fc staining in cells transduced with a non-targeting (NT) sgRNA to cells within the “CD44 High” expression gate. N) The MFI of Siglec-10-Fc staining in NT cells vs. “CD44 High” cells is shown. MFI is internally normalized to the WT cell line. O) K-562 CRISPRa cells were transduced with an sgRNA against CSPG4 and co-stained with Sig10-Fc and a CSPG4 antibody as in 4C . A representative flow cytometry plot is shown comparing Siglec-10-Fc staining in cells transduced with a non-targeting (NT) sgRNA to cells within the “CSPG4 High” expression gate. P) The MFI of Siglec-10 staining in NT cells vs. “CSPG4 High” cells is shown. MFI is internally normalized to the WT cell line. Statistical significance was determined using a two-tailed t-test. ** indicates p<0.01, * indicates p<0.05. Mean values plotted, error bars indicate SEM.

Article Snippet: The CRISPRa-v2 library (top 5 sgRNAs/gene) containing 104,535 sgRNAs was purchased from Addgene (Pooled Library 83978).

Techniques: Transduction, Selection, Staining, Flow Cytometry, Expressing, Derivative Assay, Labeling, Isolation, Two Tailed Test

Journal: bioRxiv

Article Title: Heterotypic inter-GPCR ß-arrestin coupling regulates lymphatic endothelial junctional architecture in murine lymph nodes

doi: 10.1101/435776

Figure Lengend Snippet: ( A ) Schematic of S1PR1 modulator screening system Four lentiviral vectors were transduced into U2OS cell line to enable gene activation by SAM and monitoring S1PR1 activation by TANGO system. The cells introduced with SAM sgRNA library were starved with 0.5% charcoal treated FBS, then the Venus-positive population was sorted and next-gen sequence (NGS) analysis was carried out to identify the enriched SAM sgRNA sequences. ( B ) Scatter plot showing enrichment of sgRNAs after sorting. Most sgRNAs are equally distributed in the pre-sort sample (closed gray circles) while after sorting a small fraction of sgRNAs (2,770 out of 70,290 sgRNAs) were enriched and others were not detected (open blue circles). The y-axis shows the NGS reads of sgRNAs. ( C ) Identification of top candidate genes using the MAGeCK method . The names of top ten candidate genes are indicated.

Article Snippet: The single clones were isolated from antibiotics resistant cells by limiting dilution, then introduced with the SAM sgRNA library (a gift from Feng Zhang, Addgene #1000000057) at a low multiplicity of infection.

Techniques: Activation Assay, Sequencing

Journal: eLife

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.7554/eLife.81856

Figure Lengend Snippet: ( A ) Comparison of growth phenotypes for all elements between our pilot single-sgRNA library and Horlbeck et al. data, merged by gene name (n=20,228 elements). Growth phenotypes are reported as γ (log 2 fold-enrichment of T final over T 0 , per doubling) and correlated between experiments (r=0.82). ( B ) Comparison of growth phenotypes for all elements between our pilot dual-sgRNA library and Horlbeck et al. data, merged by gene name (n=20,228 elements). Growth phenotypes are reported as γ and correlated between experiments (r=0.83). ( C ) Comparison of growth phenotypes for all elements between our pilot single- and dual-sgRNA libraries, merged by gene name (n=21,239 with 20,228 targeting elements and 1011 non-targeting elements). Growth phenotypes are reported as γ and correlated between experiments (r=0.86). ( D ) Comparison of true and false-positive rates in single element screens. ‘Positives’ (n=1363 elements) were defined as genes with a K562 CRISPRi growth screen p-value <0.001 and γ<–0.05 , and ‘negatives’ were defined as non-targeting control sgRNA pairs (n=1011 elements). ( E ) Comparison of recombination rates for non-targeting dual-sgRNA elements between replicates of our K562 growth screen. Non-targeting elements with a growth phenotype (γ>0.05 or γ<−0.05) were excluded (n=973 elements). Recombination rates were weakly correlated between replicates (r=0.30). ( f ) Comparison of recombination rates for all dual-sgRNA elements between replicates of our K562 growth screen (n=20,387 elements). Recombination rates were strongly correlated between replicates (r=0.77). ( G ) Comparison of recombination rates and growth phenotypes for all dual-sgRNA elements in our K562 growth screen (n=20,387 elements). Growth phenotypes are reported as γ. Recombination rates were strongly anticorrelated with growth phenotypes (r=−0.84).

Article Snippet: The human CRISPRi dual-sgRNA libraries with IBCs are available from Addgene (hCRISPRi_dual_1_2, Addgene #187246; hCRISPRi_dual_3_4, Addgene #187247; hCRISPRi_dual_5_6, Addgene #187248).

Techniques: Comparison, Control

Journal: eLife

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.7554/eLife.81856

Figure Lengend Snippet: ( A ) Schematics of CRISPRi transcription repressor domains and general lentiviral expression construct used for all CRISPRi effectors. UCOE = ubiquitous chromatin opening element; SFFV = spleen focus-forming virus promoter; P2A = ribosomal skipping sequence; WPRE = woodchuck hepatitis virus post-transcriptional regulatory element. Further information on repressor domains and lentiviral expression constructs can be found in the main text and Materials and methods. ( B ) Experimental design to test effects of stable expression of each CRISPRi effector on growth and transcription in K562 cells. ( C ) Growth defects of effector-expressing cells, measured as the log 2 of the ratio of mCherry-negative (effector-expressing) to mCherry-positive (not effector-expressing) cells in each well normalized to the same ratio on day 0. mCherry levels were measured for 19 days after pooling cells. Data represent mean ± SD from three independent transductions of expression constructs. p-Values are from an unpaired two-tailed t-test comparing D19 values for each sample to the D19 value for the ‘no plasmid’ sample. Average percent growth defect per day is the log 2 D19 value divided by the number of days, multiplied by 100 for a percent value. ( D ) Clustered heatmap of correlation of transcript counts from K562 cells expressing indicated CRISPRi effectors or a GFP control. Correlations across samples were calculated using normalized counts (reads per million) for all genes with mean normalized count >1 and then clustered using the Ward variance minimization algorithm implemented in scipy. r 2 is squared Pearson correlation. Data represent three independent transductions of expression constructs. ( E ) Number of differentially expressed genes ( p <0.05) for cells expressing each effector versus cells expressing GFP only. p -Values were calculated using a Wald test and corrected for multiple hypothesis testing as implemented in DeSeq2. Figure 2—source data 1. p-Values and growth defects depicted in . Figure 2—source data 2. Data depicted in .

Article Snippet: The human CRISPRi dual-sgRNA libraries with IBCs are available from Addgene (hCRISPRi_dual_1_2, Addgene #187246; hCRISPRi_dual_3_4, Addgene #187247; hCRISPRi_dual_5_6, Addgene #187248).

Techniques: Expressing, Construct, Virus, Sequencing, Two Tailed Test, Plasmid Preparation, Control

Journal: eLife

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.7554/eLife.81856

Figure Lengend Snippet: Design of constructs for CRISPR interference (CRISPRi) effector expression.

Article Snippet: The human CRISPRi dual-sgRNA libraries with IBCs are available from Addgene (hCRISPRi_dual_1_2, Addgene #187246; hCRISPRi_dual_3_4, Addgene #187247; hCRISPRi_dual_5_6, Addgene #187248).

Techniques: Construct, CRISPR, Expressing

Journal: eLife

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.7554/eLife.81856

Figure Lengend Snippet: ( A ) Experimental design to measure knockdown mediated by different CRISPR interference (CRISPRi) effectors by delivering single guide RNAs (sgRNAs) targeting either essential genes or cell surface markers. ( B ) Depletion of K562 cells expressing essential gene-targeting sgRNAs and different CRISPRi effectors, measured as the ratio of mCherry-positive (sgRNA-expressing) to mCherry-negative (not sgRNA-expressing) cells in a given well. mCherry levels were measured for 12 days after transduction, starting on day 3. Data from two replicate transductions. ( C ) Percent knockdown of cell surface markers by different CRISPRi effectors in K562 cells. Cell surface marker levels were measured on day 6 post-transduction by staining with an APC-conjugated antibody. Knockdown was calculated as the ratio of median APC signal in sgRNA-expressing cells and median APC signal in cells expressing a non-targeting control sgRNA after subtraction of background APC signal. Data from two replicate transductions. Cells expressing dCas9 and a strong CD55-targeting sgRNA are represented by a single replicate. ( D ) Distribution of anti-CD151 signal intensity (APC) in individual cells from one representative transduction. Data from second replicate are shown in . Knockdown was quantified as in C as the ratio of the median APC signals. ( E ) Percentage of cells without observable knockdown despite expressing a strong sgRNA, as quantified from the fluorescence distributions.

Article Snippet: The human CRISPRi dual-sgRNA libraries with IBCs are available from Addgene (hCRISPRi_dual_1_2, Addgene #187246; hCRISPRi_dual_3_4, Addgene #187247; hCRISPRi_dual_5_6, Addgene #187248).

Techniques: Knockdown, CRISPR, Expressing, Transduction, Marker, Staining, Control, Fluorescence

Journal: eLife

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.7554/eLife.81856

Figure Lengend Snippet: ( A ) Depletion of K562 cells expressing essential gene-targeting single guide RNAs (sgRNAs) and different CRISPRi effectors, measured as the ratio of mCherry-positive (sgRNA-expressing) to mCherry-negative (not sgRNA-expressing) cells in a given well, as in . mCherry levels were measured for 12 days after transduction, starting on day 3. Data from two replicate transductions. ( B ) Distribution of anti-CD151 signal intensity (APC) in K562 cells expressing indicated CRISPRi effectors from second replicate transduction. Knockdown was quantified as in . ( C ) Distribution of anti-CD81 signal intensity (APC) in K562 cells expressing indicated CRISPRi effectors from two replicate transductions. Knockdown was quantified as in . ( D ) Distribution of anti-CD55 signal intensity (APC) in K562 cells expressing indicated CRISPRi effectors from two replicate transductions. Cells expressing dCas9 and the CD55-targeting sgRNA are represented by a single replicate. Knockdown was quantified as in .

Article Snippet: The human CRISPRi dual-sgRNA libraries with IBCs are available from Addgene (hCRISPRi_dual_1_2, Addgene #187246; hCRISPRi_dual_3_4, Addgene #187247; hCRISPRi_dual_5_6, Addgene #187248).

Techniques: Expressing, Transduction, Knockdown

Journal: eLife

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.7554/eLife.81856

Figure Lengend Snippet: ( A ) Distribution of anti-B2M signal intensity (APC) in individual RPE1 (left) and Jurkat (right) cells expressing indicated CRISPR interference (CRISPRi) effectors and single guide RNAs (sgRNAs). Knockdown was calculated as the ratio of median APC signal in transduced (sgRNA-expressing) cells and median APC signal in non-transduced cells in the same well, after subtraction of background APC signal. ( B ) Depletion of indicated cell surface markers in HepG2 (top), HuTu-80 (middle), and HT29 (bottom) cells expressing Zim3-dCas9. Cell surface marker levels were measured 6–14 days post-transduction by staining with APC-conjugated antibodies. Knockdown was calculated as the ratio of median APC signal in sgRNA-expressing cells and median APC signal in cells expressing a non-targeting control sgRNA after subtraction of background APC signal. ( C ) Distribution of anti-B2M signal intensity (APC) in individual K562 cells expressing indicated CRISPRi effectors and sgRNAs. The Zim3-dCas9 (Hygro) cell line was generated by transduction followed by hygromycin selection and does not express a fluorescent protein. Knockdown was calculated as in A .

Article Snippet: The human CRISPRi dual-sgRNA libraries with IBCs are available from Addgene (hCRISPRi_dual_1_2, Addgene #187246; hCRISPRi_dual_3_4, Addgene #187247; hCRISPRi_dual_5_6, Addgene #187248).

Techniques: Expressing, CRISPR, Knockdown, Marker, Transduction, Staining, Control, Generated, Selection

Journal: eLife

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.7554/eLife.81856

Figure Lengend Snippet:

Article Snippet: The human CRISPRi dual-sgRNA libraries with IBCs are available from Addgene (hCRISPRi_dual_1_2, Addgene #187246; hCRISPRi_dual_3_4, Addgene #187247; hCRISPRi_dual_5_6, Addgene #187248).

Techniques: Stable Transfection, Marker, Flow Cytometry, Recombinant, Plasmid Preparation, Sequencing, Expressing, Purification, Amplification, Transfection, Software, Genome Wide

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A ) Schematic outline of the kinome shRNA and whole genome CRISPR-Cas9 screens. ( B ) Volcano plot representing gene summary of MAGeCK analysis of the kinome shRNA screens in KB2P-1.21 cells treated with olaparib. ( C ) Volcano plot representing gene summary of MAGeCK analysis of the whole genome CRISPR-Cas9 screens in RPE1-hTERT BRCA1 -/- ;p53 -/- cells treated with 2 Gy IR. ( D, E ) Growth assays in KB2P-3.4 (D) or KB1P-G3 (E) Taok1 -/- cell lines treated with the indicated doses of olaparib (D) or IR (E). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( F ) Representative images of growth assays in KB1P-G3 NT, Taok1 -/- with empty rescue construct (pOZ-empty) or Taok1 full cDNA rescue construct (pOZ-Taok1) treated with the indicated doses of olaparib. ( G ) Kaplan-Meier survival curve of mice transplanted with KB1P-4S organoids with NT or Taok1 sgRNAs and treated with olaparib. Statistical analysis was performed with the log-rank test. ( H, I ) Growth assays in KB1P-G3 Taok1 -/- cell lines complemented with indicated rescue construct. Cells were treated with indicated doses of olaparib (H) or IR (I). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: shRNA, CRISPR, Construct

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: (A) Volcano plot representing gene summary of MAGeCK analysis of the whole genome CRISPR-Cas9 screens in RPE1-hTERT BRCA1 -/- ;p53 -/- cells treated with 1 Gy IR. ( B-E ) Western blot analysis of TAOK1 protein expression of NT and TAOK1 KO in KB2P-3.4 (B), KB1P-G3 including HA-tagged, Taok1 cDNA rescue (pOZ-Taok1) (C), MDA-MB-436 (D) and KB2P-1.21 (E) cell lines. ( F ) TIDE analysis of polyclonal KB2P-1.21 cells. ( G, H ) Growth assays in KB1P-G3 NT, Taok1 -/- and Taok1 rescue cells treated with the indicated doses of olaparib (G) or talazoparib (H). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( I, J ) Growth assays in MDA-MB-436 NT and TAOK1 -/- cell lines treated with indicated doses of olaparib (I) or IR (J). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( K, L ) Growth assays in KB2P-1.21 NT and Taok1 sgRNA cells treated with the indicated doses of olaparib (K) or IR (L). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( M ) TIDE analysis of polyclonal KB1P-4S organoids. ( N ) Immunohistochemistry staining for TAOK1 of untreated BRCA1;p53-deficient mouse mammary tumors with NT or Taok1 sgRNA. ( O ) Olaparib response of KB1P-4S organoids transduced with NT or Taok1 sgRNAs. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: CRISPR, Western Blot, Expressing, Immunohistochemistry, Staining, Transduction

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A-C ) Quantification (A, B) and representative images (C) of growth assays in KB1P-G3B1 WT (A) and KB1P-G3 WT and Taok1 -/- cells treated with the indicated doses of CP 43 and olaparib. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( D ) Western blot analysis of expression level of HA-tagged Taok1 rescue constructs. ( E ) In vitro kinase assay of indicated KB1P-G3 cell lines. Protein was isolated from KB1P-G3 cell lines by co-IP with HA-tag. NT cell line was used as negative control with no HA-tag while the other cell lines expressed an HA-tag with the indicated construct. Bars represent mean ± SD, statistical analysis was done with two-way ANOVA followed by Dunnett’s test. ( F, G ) Representative images of growth assay of KB1P-G3 NT, Taok1 -/- and Taok1 full cDNA rescue (pOZ-Taok1) or two different kinase mutants (pOZ-K57A, pOZ-D169A) and a double mutant (pOZ-K57A+D169A) treated with indicated doses of olaparib (F) or IR (G).

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Western Blot, Expressing, Construct, In Vitro, Kinase Assay, Isolation, Co-Immunoprecipitation Assay, Negative Control, Growth Assay, Mutagenesis

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A ) Graph representing percentage of cells with micronuclei upon 0.5 µM olaparib treatment for 48 h. Bars are plotted as mean ± SD, statistical analysis was performed using ANOVA followed by Tukey’s multiple comparison test. ( B ) Quantification of RAD51 foci formation upon 10 µM olaparib. Bars represent mean ± SD, statistical analysis was performed using ANOVA followed by Tukey’s multiple comparison test. ( C ) Representative images of RAD51 foci formation upon 10 µM olaparib in Brca1 reconstituted KB1P-G3B1, KB1P-G3 and KB2P-3.4 cell lines with the indicated modifications. ( D ) Quantification of DSB spectrum assay in HEK293T cells treated with the indicated siRNAs. Each dot represents an experiment ran in duplicates, bars represent mean ± SD, statistical analysis was performed using ANOVA followed by Tukey’s multiple comparison test. ( E ) Growth assays of KB1P-G3 NT, Taok1 -/- and TAOK1 rescue cells treated with the indicated doses of cisplatin. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Comparison

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A ) Quantification of RAD51 foci formation 3 h post 10 Gy IR. Bars represent mean ± SD, statistical analysis was performed using ANOVA followed by Tukey’s multiple comparison test. ( B ) Verification of siRNA KD efficiency in HEK293T DSB-Spectrum_V1 cell line. ( C ) Growth assays with MDA-MB-436 NT and TAOK1 KO cell lines treated with the indicated doses of cisplatin. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( D, E ) Growth assays in KB1P-G3 NT, TAOK1 KO and Taok1 rescue cell lines treated with indicated doses of carboplatin (D) or oxaliplatin (E). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Comparison

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: (A) Representative images of IHC staining for TAOK1 on human tumor samples of prostate and lung showing variable nuclear and diffuse cytoplasmatic localization. ( B ) Representative images of IHC staining for TAOK1 on human tumor samples of prostate, bladder and ovary showing varying TAOK1 expression levels. ( C )Western blot analysis of KB1P-G3 NT samples of cytoplasmatic (C) and nuclear (N) fraction and Taok1 -/- whole cell lysate. ( D ) Representative images of immunofluorescence for cellular expression pattern of KB1P-G3 Taok1 -/- cells with HA-tagged pOZ-Taok1 rescue construct compared to pOZ-empty vector control. ( E ) Growth assay in KB1P-G3 NT, Taok1 -/- and Taok1 rescue cell lines treated with indicated doses of camptothecin. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( F ) Growth assay in KB2P-3.4 NT and Taok1 -/- clones treated with indicated doses of JH-RE-06 with or without olaparib (0.1 µM). Statistical analysis was performed using two-way ANOVA followed by Tukey’s test. ( G ) DNA fiber analysis in indicated cell lines with or without 4 mM HU treatment (3 h). Bar represents mean of IdU/CldU ratio of at least 250 fibers, statistical analysis was done with ANOVA followed by Tukey’s multiple comparison test. (H) DNA fiber analysis in indicated cell lines treated with 10 µM olaparib 2h prior and while labelling with CldU and IdU, ssDNA was digested with S1 nuclease if indicated. Bar represents the mean of IdU track length of at least 250 fibers, statistical analysis was done with ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Immunohistochemistry, Expressing, Western Blot, Immunofluorescence, Construct, Plasmid Preparation, Control, Growth Assay, Clone Assay, Comparison

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A ) Quantification of SIRF foci formation of HA-tagged TAOK1 with and without 2 mM HU. Bars represent mean ± SD, statistical analysis was performed using ANOVA followed by Tukey’s multiple comparison test. ( B ) Representative images of analysis shown in ( A ). ( C ) Volcano plot of mass spectrum analysis of TAOK1 co-IP samples from KB1P-G3 NT nuclear fraction compared to TAOK1 KO. Positive log2 fold change values indicate enrichment in the NT nuclear fraction compared to TAOK1 KO of four independent replicas. ( D ) Western blot of input and co-IP of KB1P-G3 NT and TAOK1 KO. Pulldown was performed with PCNA antibody. ( E ) Growth assay of KB1P-G3 NT, TAOK1 KO and TAOK1 rescue cells treated with topotecan at indicated doses. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s test. ( F ) Growth assay of KB1P-G3 NT, TAOK1 KO and TAOK1 rescue cells treated with JH-RE-06 alone or in combination with olaparib. Statistical analysis was performed using two-way ANOVA followed by Tukey’s test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Comparison, Co-Immunoprecipitation Assay, Western Blot, Growth Assay

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A ) Graph showing replication speed by total track length of untreated DNA fiber assay in KB1P-G3 NT and TAOK1 KO cells. Bar represents the mean of at least 250 fibers, statistical analysis was done with unpaired t-test. ( B ) DNA fiber analysis in indicated cell lines with or without 4 mM HU treatment. Bar represents the mean of IdU/CldU ratio of at least 300 fibers, statistical analysis was done with unpaired t-test. ( C ) DNA fiber analysis in indicated cell lines treated with 10 µM olaparib 2h prior and while labelling with CldU and IdU, ssDNA was digested with S1 nuclease if indicated. Bar represents the mean of IdU track length of at least 250 fibers, statistical analysis was done with ANOVA followed by Tukey’s multiple comparison test. ( D, E ) Quantification (D) and representative images (E) of immunofluorescence analysis of RPA foci formation in KB1P-G3 NT, TAOK1 KO and TAOK1 rescue cells upon olaparib treatment (10 µM for 16 h). Bars indicate the mean number of foci per nucleus of at least 1000 nuclei. Statistical analysis was done with ANOVA followed by Tukey’s multiple comparison test. (F) Western blot of input and co-IP of KB1P-G3 NT and TAOK1 rescue cells. Pulldown was performed with TRIM25 antibody. ( G, H ) Western blot of indicated KB1P-G3 (H) and MDA-MB-436 (I) cell lines for ISG15 protein levels. Cells were untreated or treated with 10 µM olaparib for 24 h. Vinculin was used as loading control. ( I, J ) Schematic predicting replication dynamics in presence (I) or absence of TAOK1 (J) in BRCA1/2-deficient cells. (I) TAOK1 interacts with PCNA and TRIM25 and does not allow alternative fork protection mechanisms or ssDNA gap repair in BRCA-deficient cells, leading to RF instability. (J) In the absence of TAOK1, increased ISG15 expression and potentially recruitment to RF activates fork protection and gap suppression mechanisms and thus, RF stability.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Comparison, Immunofluorescence, Western Blot, Co-Immunoprecipitation Assay, Control, Expressing

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A, B ) Representative images (A) and quantification (B) of immunofluorescence analysis of RPA foci formation in KB2P-3.4 NT and Taok1 -/- cells upon olaparib treatment (10 µM for 16 h). Bars indicate mean number of foci per nucleus of at least 1000 nuclei. Statistical analysis was done with ANOVA followed by Tukey’s multiple comparison test. ( C, D ) Graph representing ssDNA intensity of native BrdU incorporation in KB1P-G3 (C) and MDA-MB-436 (D) cell lines. Cells were treated with 0.75 µM olaparib for 5 h. Ordinary one-way ANOVA with Dunnett’s multiple comparison test was used for statistical analysis. ( E ) Representative images of PLA assay between TRIM25 and HA-tag in KB1P-G3 NT and Taok1 rescue cells. ( F ) Kaplan-Meier curve of survival probability of SCAN-B dataset treated with non-chemotherapy. Statistical analysis was done with log-rank test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Immunofluorescence, Comparison, BrdU Incorporation Assay

Journal: bioRxiv

Article Title: TAOK1 regulates chemo- and radiosensitivity in BRCA1/2-deficient tumors

doi: 10.1101/2025.10.14.682031

Figure Lengend Snippet: ( A, B ) Analysis of the SCAN-B dataset for TAOK1 high or low expression. Kaplan-Meier curves for survival probability of all patients (A) and patients treated with chemotherapy specifically (B). Risk tables are shown below. Statistical analysis was done with log-rank test.

Article Snippet: For CRISPR-Cas9 editing of human MDA-MB-436 cell line, NT sgRNA and TAOK1 sgRNAs were cloned into pX330 vector (Addgene, #42230) with puromycin resistance cassette.

Techniques: Expressing