Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Ter119 negative cells and Ter119 positive erythroid cells were purified from wild-type mouse bone marrow cells. G4 levels were tested by flow cytometry using the BG4 antibody that specifically recognizes G4. Quantification is on the right. (B) Bone marrow lineage-negative cells were cultured in Epo medium for 2 days. G4 levels were tested on different days using flow cytometry by the BG4 antibody. Quantification is on the right. (C) CD34+ human HSPCs were cultured in Epo medium for 21 days. The levels of G4 were measured by flow cytometry as in B at the indicated time. Cells at day 7, 14, and 21 represent proerythroblasts, polychromatic to orthochromatic erythroblasts, and orthochromatic to mature red blood cells, respectively. (D) Flow cytometric assays of G4 levels in the indicated bone marrow lineage cells purified from wild-type mice. (E) Quantification of D. (F) Gating strategy of various erythroblasts. Populations I to VI represent proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts, late orthochromatic to reticulocytes, and mature red blood cells, respectively. (G-H) Flow cytometric assay of G4 level in bone marrow erythroid populations I (G) and V (H) from the indicated mice. Quantification is on the right. (I) Bone marrow lineage negative cells from the indicated mice were cultured in Epo medium for 2 days. G4 levels on different days were measured by flow cytometry using BG4 antibody. Quantification is below the histogram. (J) CD34+ cells were transduced with lentiviral vectors expressing indicated sgRNAs and Cas9. Cells were then harvested for Western blotting of the indicated proteins at day 9 in culture. (K) Quantitative analyses of G4 levels in cells from J using flow cytometric assays. (L) Quantitative analyses of cell death in cells from J using flow cytometric assays. The dead cells are defined as propidium iodide and annexin V double positive. (M) Quantitative analyses of G4 levels in bone marrow mononuclear cells from the patient with DDX41 mutated MDS. All the error bars represent the SEM of the mean. The comparison between two groups was evaluated with 2 tailed t tests, and the comparison among multiple groups was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. ns: not significant.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Purification, Flow Cytometry, Cell Culture, Transduction, Expressing, Western Blot, Comparison

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Epo medium-cultured mouse bone marrow lineage negative HSPCs were treated with 1 μM PDS for the indicated time. Immunofluorescence assays of γ-H2AX were performed, and representative images of the erythroid cells were presented. Scale bar: 5 μm. (B) Flow cytometry assay of the cells in A. (C) Statistical quantification of γH2AX signals in B. (D) Epo medium-cultured mouse bone marrow lineage negative HSPCs were cultured for 1 day, followed by the treatment of 1 μM PDS for 6 hours. Quantitative RT-PCR analyses of indicated ribosome RNAs were performed using different primer sets. (E) Western blotting assays of indicated in cells from D. Actin was used as a loading control. (F) Same as D except that bone marrow lineage negative HSPCs from HBBCre:Ddx41 fl/fl mouse were cultured for 1 day before the quantitative RT-PCR assays. (G) Western blotting assays of the indicated proteins in F. Cells from both day 1 and day 2 cultured cells were analyzed. (H) CD34+ cells were transduced with lentiviral vectors expressing indicated sgRNAs and Cas9. Cells were then harvested for Western blotting of the indicated proteins at day 9 in culture. (I) Immunohistochemical stains of p53 in bone marrow core biopsies from the patient in normal individual. Scale bar: 100 μm. (J) Quantification of γ-H2AX in bone marrow mononuclear cells from the patient in I and 2 control individuals. All the error bars represent the SEM of the mean. The comparison between two groups was evaluated with 2 tailed t tests, and the comparison among multiple groups was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01, ns: not significant.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Cell Culture, Immunofluorescence, Flow Cytometry, Quantitative RT-PCR, Western Blot, Control, Transduction, Expressing, Immunohistochemical staining, Comparison

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Representative wide-field picture and H&E stains of bone marrow organoid in culture. (B) Whole-mount 3D imaging of the organoids. Imaris was used for cell surface rendering. Organoids were stained with indicated antibodies and subsequently imaged using a laser scanning confocal platform. (C) Confocal immunofluorescence assays of erythroid islands in the iPSC-derived bone marrow organoids (left) and a primary human bone marrow biopsy (right). CD71 was labeled with green for organoids and magenta for primary bone marrow. DAPI: blue. (D) Flow cytometry assays of the organoids using indicated antibodies for various lineages. (E) 10,000 CellVue-labeled donor CD34+ HSPCs were co-incubated with iPSC-derived bone marrow organoids for 3 days in each well of a 96-well plate, followed by an immunofluorescence assay. Representative pictures show the engraftment of donor hematopoietic cells into the organoid. Green, red, and blue represent CD71, CellVue, and DAPI-positive nuclei, respectively. The arrow points to an engrafted CellVue positive cell expressing CD71. (F) Flow cytometry of the organoids using indicated antibodies for various lineages of the engrafted cells in organoids from E. (G) Same as E, except the donor CD34+ cells were transduced with lentiviral vectors expressing Cas9 and indicated sgRNAs before co-incubation. After 3 days, the cells were collected for flow cytometric assays of erythroid and myeloid differentiation of CellVue-positive donor hematopoietic cells and negative iPSC-derived hematopoietic cells. Each data point represents cells combined from 10 organoids. The comparison was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01. (H) Schematic model of the function of DDX41 during erythropoiesis. The diagram is generated through BioRender.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Imaging, Staining, Immunofluorescence, Derivative Assay, Labeling, Flow Cytometry, Incubation, Expressing, Transduction, Comparison, Generated

Journal: bioRxiv

Article Title: dnaudit + pydnaweb: A lightweight text-based planning and documentation workflow for genetic cloning with automatic verification

doi: 10.1101/2025.05.31.657172

Figure Lengend Snippet: A . amplification of Fragment A by PCR; B . amplification of Fragment B containing the gRNA by PCR; C . amplification of Fragment C by fusion PCR; D . digestion of the Cas9 plasmid using the MssI restriction enzyme to generate the final Cas9 cassette; E . amplification of the dDNA cassette (GFP) by PCR; F . integration of the Cas9 cassette and Fragment C in the C. albicans HIS1 locus by homologous recombination.

Article Snippet: Finally, Fragment C was co-transformed into C. albicans SC5314 together with the appropriately digested CAS9 pADH99 plasmid (with MssI restriction enzyme; Addgene plasmid # 90979; ) and the donor DNA. dDNAs ( ) were amplified from the template CIp10–γmGFP (Addgene plasmid # 163119, 27) using the oligonucleotides dDNA_ATO1_GFP_Fw and dDNA_ATO1_GFP_Rv as forward and reverse primers, respectively ( Table S1 ).

Techniques: Amplification, Plasmid Preparation, Homologous Recombination

Journal: bioRxiv

Article Title: dnaudit + pydnaweb: A lightweight text-based planning and documentation workflow for genetic cloning with automatic verification

doi: 10.1101/2025.05.31.657172

Figure Lengend Snippet: The Cas9 endonuclease is guided by the gRNA to the target site within the ATO1 gene. The gRNA binds to the complementary DNA sequence, allowing Cas9 to introduce a double-strand break (DSB) at that locus (Figure S2). The donor DNA cassette containing the gene encoding GFP facilitates homology-directed repair (HDR), resulting in the insertion of the GFP sequence into the 3’ end of the ATO1 gene.

Article Snippet: Finally, Fragment C was co-transformed into C. albicans SC5314 together with the appropriately digested CAS9 pADH99 plasmid (with MssI restriction enzyme; Addgene plasmid # 90979; ) and the donor DNA. dDNAs ( ) were amplified from the template CIp10–γmGFP (Addgene plasmid # 163119, 27) using the oligonucleotides dDNA_ATO1_GFP_Fw and dDNA_ATO1_GFP_Rv as forward and reverse primers, respectively ( Table S1 ).

Techniques: Sequencing, Introduce

Journal: bioRxiv

Article Title: CRISPR disruption and UK Biobank analysis of a highly conserved polymorphic enhancer suggests a role in male anxiety and ethanol intake

doi: 10.1101/572065

Figure Lengend Snippet: A and C , Dark field images of 10µm sections through (A) the hypothalamic region and (C) the amygdala region of wildtype mouse probed with S labelled Gal antisense RNA. (B and D) vibratome sections through (B) the hypothalamic and (D) the amygdala region of a mouse transgenic for the human GAL5.1-LacZ reporter construct (hGAL5.1-LacZ) demonstrating LacZ expression in cells of the periventricular (PVN) Dorsomedial (DMH) and arcuate (ARC) nuclei (V3, 3 rd ventricle) of the hypothalamus (A and B) and the (MeA) medial amygdaloid nucleus of the amygdala (C and D). E. Comparative genomic analysis of sequence flanking the human GAL locus demonstrating the position (numbered black scale bar at top), distance (blue arrows) and depth of conservation (green peaks) of the human GAL5.1 locus (red box) relative to the GAL gene (blue box). F and G. Sequence data derived from PCR products of ear clip DNA derived from a homozygote GAL5.1 KO mouse (thick black bar labelled GAL5.1KO58) blasted against the mouse genome (UCSC browser). The sequence disruption produced by introduction of CAS9/sgRNA is displayed relative to the PAM sequence (green box) of each sgRNA demonstrates the accuracy of (F) 5sgRNA and ( G) 3sgRNA relative to the missing regions of the genome (thin black lines and chevrons). Electrophoresis gels demonstrating the difference in PCR product sizes are published elsewhere (Hay et al. , 2018).

Article Snippet: sgRNA molecules were microinjected at a concentration 10 ng/ µ l each into the cytoplasm of 1-cell C57/BL6 embryos (Charles River) as described together with 10 ng/ µ l CAS9 mRNA (Life Technologies).

Techniques: Transgenic Assay, Construct, Expressing, Sequencing, Derivative Assay, Produced, Electrophoresis