Journal: bioRxiv

Article Title: Endogenous gene tagging with FnCas9 to track and sort neural lineages from 3D cortical organoids

doi: 10.1101/2025.01.09.631995

Figure Lengend Snippet: schematic of the process that was optimized for FnCas9-based HDR-mediated endogenous tagging of the selected marker gene, DCX. The linearized donor DNA template used for knock-in is shown with various components, along with left and right homology arms complementary to the C-terminus end of the DCX . The agarose gel shows PCR genotyping of the antibiotic-selected cells with the successful integration of the DNA donor template. (NTC, no DNA template control)

Article Snippet: The plasmid containing FnCas9 Addgene 130969 ( ) was incorporated with a crRNA sequence targeting the DCX C-terminus at the gRNA scaffold and was confirmed with PCR genotyping as well as Sanger sequencing.

Techniques: Marker, Knock-In, Agarose Gel Electrophoresis, Control

Journal: bioRxiv

Article Title: Endogenous gene tagging with FnCas9 to track and sort neural lineages from 3D cortical organoids

doi: 10.1101/2025.01.09.631995

Figure Lengend Snippet: a) Schematic of the protocol optimized for the development of cortical organoids from the FnCas9-tagged hESCs (details in the “Methods” section). b) Left panel: expression of Ki67 and SOX2, highlighting the neural rosette cells and different developmental stages of cortical organoids (hCOs) at days 25, 52, and 75. Right panel: co-immunostaining of the organoid sections with neural marker proteins at days 25 and 60 showing their maturation (scale: 50 µm) c) Immunofluorescence-based co-localization of DCX and tdTomato expression within the cells dissociated from DCX-tdTomato-hCOs. (scale: 25 µm) d) The colocalization between the signals obtained from Red: Green channels are quantified as tM (thresholded Mander’s split colocalization coefficient), n = 30, error bars represent S.E.M., a single-mean two-tailed unpaired t-test is applied, and p values of **** ≤ 0.0001 (values from independent measurements are represented as dots). e) Real-time FACS-based cell sorting of live cells from DCX-tdTomato-tagged hCOs and unedited hCOs at day 52 (representative of three independent experiments).

Article Snippet: The plasmid containing FnCas9 Addgene 130969 ( ) was incorporated with a crRNA sequence targeting the DCX C-terminus at the gRNA scaffold and was confirmed with PCR genotyping as well as Sanger sequencing.

Techniques: Expressing, Immunostaining, Marker, Immunofluorescence, Two Tailed Test, FACS

Journal: EMBO Reports

Article Title: Global and precise identification of functional miRNA targets in mESCs by integrative analysis

doi: 10.15252/embr.202254762

Figure Lengend Snippet: Immunoblot analysis of DROSHA, DICER, AGO1, and AGO2 in WT and their respective KO mESC lines. TUBULIN was used as a loading control. Representative blot of three independent experiments is shown. PCA of gene expression as measured by RNA sequencing in miRNA _KO and WT samples. Biological replicates are indicated with corresponding colors. MA plots of the DGE analysis in miRNA _KO mutants versus WT. Significant up‐ and downregulated genes are colored in red and blue, respectively. Overlap of up‐ (left) and downregulated (right) genes in miRNA _KO mESCs. Comparison of miRNA loading for small RNA sequencing (Input) versus Argonaute2 & 1 (AGOs) RNA immunoprecipitation (RIP) and sequencing for all miRNAs. MiRNAs that show statistically significant difference between the two measured (adjusted P ‐value < 0.1) are highlighted. Data information: In (C and E), significance in differential analysis was determined using an adjusted P ‐value threshold obtained from DESeq2 of 0.1. In (D), an adjusted P ‐value threshold of 0.2 was applied. Source data are available online for this figure.

Article Snippet: pX458‐sgRNA_ Ago1 _3 , Addgene , #73535.

Techniques: Western Blot, Control, Gene Expression, RNA Sequencing, Comparison, RNA Immunoprecipitation, Sequencing

Journal: EMBO Reports

Article Title: Global and precise identification of functional miRNA targets in mESCs by integrative analysis

doi: 10.15252/embr.202254762

Figure Lengend Snippet:

Article Snippet: pX458‐sgRNA_ Ago1 _3 , Addgene , #73535.

Techniques: Recombinant