Journal: bioRxiv

Article Title: TET2 lesions enhance the aggressiveness of CEBPA- mutant AML by rebalancing GATA2 expression

doi: 10.1101/2023.03.28.534511

Figure Lengend Snippet: ( A ) Schematic representation of generation of Tet2 -knockout clones with CRISPR/Cas9. ( B ) Proliferative outgrowth of Cebpa p30/p30 cells with Tet2 indels. ( C ) Volcano plot depicting differentially expressed genes dependent on the Tet2 mutational status in Cebpa p30/p30 cells (n=5–7 per group). ( D ) Experimental setup for evaluating the effect of Tet2 -deficiency ( Tet2 −/− ) in Cebpa DM AML initiation in vivo . ( E ) Myeloid (Mac1 + ) contribution of donor-derived blood and bone marrow (BM) cells evaluated at 12, 24, and 36 weeks after BM transplantation and Cre-LoxP recombination to generate a Cebpa −/p30 and Tet2 −/− hematopoietic compartment (n=3–6 per genotype and timepoint). ( F ) Survival of lethally irradiated recipient mice after BM transplantation and Cre-LoxP recombination (n=12–14/group). ( G ) Volcano plot depicting differentially expressed genes dependent on Tet2 deficiency status in Cebpa −/p30 leukemic blasts (n=3 per group). ( H ) Frequency of proliferating (Ki67 + ) cells in BM of moribund recipient mice (n=3 per group). **=P<0.01, ***=P<0.001, ****=P<0.0001

Article Snippet: For generation of Tet2 or Gata2 mutated clones, Cebpa p30/p30 cells were electroporated with ribonucleoparticles containing recombinant Cas9 nuclease from Streptococcus pyogenes (Sp) (#1081058, IDT), tracrRNA (#1075927, IDT) and crRNAs (Alt-R® CRISPR-Cas9 crRNA, IDT) targeting Tet2 and Gata2 , respectively. crRNAs were designed using the CHOPCHOP web tool (chopchop.cbu.uib.no) ( Supplemental table 4 ). crRNA and tracrRNA molecules were complexed at room temperature and assembled with recombinant SpCas9 according to manufacturer’s protocols (IDT).

Techniques: Knock-Out, Clone Assay, CRISPR, In Vivo, Derivative Assay, Transplantation Assay, Irradiation

Journal: bioRxiv

Article Title: TET2 lesions enhance the aggressiveness of CEBPA- mutant AML by rebalancing GATA2 expression

doi: 10.1101/2023.03.28.534511

Figure Lengend Snippet: ( A ) Experimental setup for evaluating the effect of Gata2 knockdown, via short hairpin RNA (shRNA) mediated silencing, on Cebpa p30/p30 leukemic cells in a competitive in vivo assay. ( B ) Gata2 mRNA in Cebpa p30/p30 leukemic cells prior to transplantation. ( C ) Representative flow cytometry profiles of input and output of shControl (no knockdown), sh Gata2 A (low knockdown), and sh Gata2 D (high knockdown). ( D ) Competitive advantage of targeting shRNA (GFP + ) vs. non-targeting shRNA (YFP + ) cells in vivo assessed as by flow cytometry (n=3–4 per group). ( E ) Experimental setup for Gata2 CRISPR/Cas9 mutagenesis in Cebpa p30/p30 cells, and outgrowth of heterozygous mutated clones. Percentages of Gata2 mutated clones are indicated. ( F ) Growth curve of Cebpa p30/p30 clones with Gata2 mutation ( Cebpa p30/p30 Gata2 +/MUT ) or wild type Gata2 ( Cebpa p30/p30 Gata2 +/+ ). Red lines mark individual clones. (G) Presence or absence of GATA2 mutations ( GATA2 MUT ) in CEBPA double mutated ( CEBPA DM ) AML cases (n=460) with or without TET2 mutations ( TET2 MUT ) in aggregated data from published cohorts – , , , . *=P<0.05, **=P<0.01

Article Snippet: For generation of Tet2 or Gata2 mutated clones, Cebpa p30/p30 cells were electroporated with ribonucleoparticles containing recombinant Cas9 nuclease from Streptococcus pyogenes (Sp) (#1081058, IDT), tracrRNA (#1075927, IDT) and crRNAs (Alt-R® CRISPR-Cas9 crRNA, IDT) targeting Tet2 and Gata2 , respectively. crRNAs were designed using the CHOPCHOP web tool (chopchop.cbu.uib.no) ( Supplemental table 4 ). crRNA and tracrRNA molecules were complexed at room temperature and assembled with recombinant SpCas9 according to manufacturer’s protocols (IDT).

Techniques: Knockdown, shRNA, In Vivo, Transplantation Assay, Flow Cytometry, CRISPR, Mutagenesis, Clone Assay

Journal: bioRxiv

Article Title: TET2 lesions enhance the aggressiveness of CEBPA- mutant AML by rebalancing GATA2 expression

doi: 10.1101/2023.03.28.534511

Figure Lengend Snippet: ( A ) Gata2 mRNA expression in mouse Cebpa p30/p30 leukemic granulocyte/monocyte progenitors (GMPs) vs normal GMPs and, ( B ) CEBPA binding to the Gata2 distal hematopoietic enhancer ( G2 DHE; −77kb) region, data from Jakobsen et al. (n=2–4 per group). ( C ) Schematic genomic view of the Gata2 distal hemeatopoietic enhancer ( G2 DHE), including tracks from CEBPA and H3K27Ac chromatin immunoprecipitation sequencing (ChIP-seq) in Cebpa p /p30 cells (data from Heyes et al. ), TET2 ChIP-seq in AML-ETO expressing cells (data from Rasmussen et al. ),Targeting of the G2 DHE by dual-( D ) or single-( E ) guided CRISPR-Cas9 in Cebpa p30/p30 cells using indicated sgRNAs (n=3/condition). ( F ) Experimental setup for evaluating the effects of Cebpa knockout on Gata2 V2 mRNA expression and DNA methylation of the CpG island at the promoter of Gata2 V2 in MLL-fusion driven AML ( iMLL-AF9 ). ( G ) Cebpa and ( H ) Gata2 V2 mRNA expression upon induction of Cre-LoxP recombination and, ( I ) DNA methylation of the Gata2 V2 promoter CpG-island (2 biological replicates per genotype). ( J ) Frequency of GATA2 and/or TET2 mutations ( GATA2 MUT and TET2 MUT , respectively) in CEBPA high expressing ( CEBPA HIGH n=45) vs. CEBPA low expressing (CEBPA LOW n=61) AML cases, data from Beat AML cohort . *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001

Article Snippet: For generation of Tet2 or Gata2 mutated clones, Cebpa p30/p30 cells were electroporated with ribonucleoparticles containing recombinant Cas9 nuclease from Streptococcus pyogenes (Sp) (#1081058, IDT), tracrRNA (#1075927, IDT) and crRNAs (Alt-R® CRISPR-Cas9 crRNA, IDT) targeting Tet2 and Gata2 , respectively. crRNAs were designed using the CHOPCHOP web tool (chopchop.cbu.uib.no) ( Supplemental table 4 ). crRNA and tracrRNA molecules were complexed at room temperature and assembled with recombinant SpCas9 according to manufacturer’s protocols (IDT).

Techniques: Expressing, Binding Assay, ChIP-sequencing, CRISPR, Knock-Out, DNA Methylation Assay

Journal: Cell Reports

Article Title: Rare Variant Burden Analysis within Enhancers Identifies CAV1 as an ALS Risk Gene

doi: 10.1016/j.celrep.2020.108456

Figure Lengend Snippet:

Article Snippet: CRISPR/Cas9 Ribonucleoproteins were formed by complexing 240ng gRNA duplex with 1250ng Alt-R V3 Cas9 Protein (IDT) in 10 μL buffer R (from 10 μL Neon transfection kit, ThermoFisher Scientific) - a 1:1 molar ratio - for 10 minutes.

Techniques: Recombinant, Electroporation, Modification, SYBR Green Assay, Random Hexamer Labeling, Protease Inhibitor, Western Blot, Plasmid Preparation, BIA-KA, Transfection, CRISPR, Software, Imaging

Journal: bioRxiv

Article Title: Aging restricts maturation of CXCL13 + T follicular helper cells in human immunity

doi: 10.64898/2026.04.03.715992

Figure Lengend Snippet: a, Schematics of key differences in Tfh cell maturation between humans (left) and mice (right). b, Gene expression signatures of all CD4 + T cell clusters from tonsillar T cell scRNA-seq. c, Volcano plot showing differential gene expression of GC-Tfh cells (left) vs. CXCL13 + GC-Tfh cells (right). d, Select subset-specific marker gene expression in Tfh, GC-Tfh, and CXCL13 + GC-Tfh cells. e, Schematic overview for assessment of CXCL13 production by non-, pre-, and GC-Tfh cells. f, Exemplary gating for non-Tfh (PD-1 - CXCR5 - ), pre-Tfh (PD-1 lo CXCR5 lo ), and GC-Tfh cells (PD-1 hi CXCR5 hi ). g, CXCL13 secretion by different sorted T cell subtypes cultured with or without B cells (n = 4). h, Schematic representation for CRISPR/Cas9-based editing of tonsil samples. i, CXCL13 ELISA of tonsillar cell culture supernatants for indicated transcription factor knockouts vs. scramble control (n = 14; ≤ 30 yo). Data were analyzed using two-way ANOVA with Šídák’s multiple comparison test ( g ) and one-way ANOVA with Dunnett’s multiple comparisons test ( i ). * P < 0.05, ** P < 0.01.

Article Snippet: Editing efficiencies ( Table S3 ) were calculated using the Inference of CRISPR Edits (ICE) analysis tool (Synthego Performance Analysis 2019, v3.0) to ensure target gene knockout.

Techniques: Gene Expression, Marker, Cell Culture, CRISPR, Enzyme-linked Immunosorbent Assay, Control, Comparison

Journal: bioRxiv

Article Title: Aging restricts maturation of CXCL13 + T follicular helper cells in human immunity

doi: 10.64898/2026.04.03.715992

Figure Lengend Snippet: a, Number of differentially expressed genes (DEGs, >50yo vs. <30yo) over pseudotime during Tfh cell development from naïve CD4 + T cells (left) to CXCL13 + GC-Tfh cells (right). False discovery rate (FDR) is set to < 0.05. b, Volcano plot showing differential gene expression of Tfh cells from older (left) vs. younger donors (right). c, Schematic representation for CRISPR/Cas9-based editing of naïve CD4 + T cells followed by T cell polarization and flow cytometric analysis. d, Expression of CXCR5, FOXP3, and PD-1 in naïve CD4 + T cells cultured under Tfh-polarizing conditions (dark grey) or non-polarizing conditions (light grey). e, Log2 fold change relative to matched scramble controls for Th1 (left) and Th17 (right) cell frequency in TF-knockouts under Th1- or Th17-polarizing conditions. f, Log2 fold change relative to matched scramble controls for Tfh cell frequency in TF-knockouts under Tfh-polarizing conditions (gated on CD4 + CD19 - FOXP3 - CXCR5 + PD-1 + cells). Data were analyzed using a mixed effects model and Dunnett’s multiple comparisons test ( f ), one sample t test ( e ). Error bars SEM ( e-f ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Editing efficiencies ( Table S3 ) were calculated using the Inference of CRISPR Edits (ICE) analysis tool (Synthego Performance Analysis 2019, v3.0) to ensure target gene knockout.

Techniques: Gene Expression, CRISPR, Expressing, Cell Culture

Journal: Frontiers in Genome Editing

Article Title: Genome Editing With TALEN, CRISPR-Cas9 and CRISPR-Cas12a in Combination With AAV6 Homology Donor Restores T Cell Function for XLP

doi: 10.3389/fgeed.2022.828489

Figure Lengend Snippet: Assessment of on and off-target TALEN-, Cas9-and Cas12a-mediated activity. (A) Schematic diagram of the SH2D1A locus showing the start of the protein coding region of exon 1, TALEN-L and -R binding sites (orange), Cas9 (green) and Cas12a (blue) gRNA targets (B) T cell genome editing experimental timeline (C) Healthy control (HC) T cells nucleofected with TALEN mRNA, Cas9 or Cas12a RNPs, assessed for SAP expression by intracellular SAP staining and flow cytometry at d6 post nucleofection (n = 3–6, mean, SEM) (D) % INDEL frequency by TIDE analysis of sanger sequencing data of PCR amplicons amplified from nuclease treated T cell genomic DNA (n = 3–7, mean, SEM) (E–G) NGS-generated data of modifications at on target (ON) and in silico predicted off-target loci (OT1-10) for [E] TALEN [F] Cas12a-1 and [G] Cas9-3 nucleases (n = 1, treated (Tr) vs. untreated (UT), OT sites marked * p < 0.0001).

Article Snippet: Stimulated PBMCs were nucleofected with either in vitro -transcribed TALEN mRNA, or ribonucleoprotein (RNP) complexes of Cas9 (Alt-R ® S. p.HiFi Cas9 V3, IDT) or Cas12a (Alt-R ® AsCas12a Ultra, IDT) protein and their respective guides, according to the timeline ( ).

Techniques: Activity Assay, Binding Assay, Control, Expressing, Staining, Flow Cytometry, Sequencing, Amplification, Generated, In Silico

Journal: Frontiers in Genome Editing

Article Title: Genome Editing With TALEN, CRISPR-Cas9 and CRISPR-Cas12a in Combination With AAV6 Homology Donor Restores T Cell Function for XLP

doi: 10.3389/fgeed.2022.828489

Figure Lengend Snippet: Serum free AAV6 transduction maintains HDR efficiency at reduced MOI. (A–C) %GFP + T cells transduced with G7bc AAV in HS or HS-free conditions 15 min post-nucleofection with (A) TALEN, (B) Cas12a-1 RNP or (C) Cas9-3 RNP (N = 2, mean, SD). (D) %GFP + T cells transduced with G7bc AAV at a range of MOIs 2h prior to TALEN nucleofection, in human serum (HS) or HS-free transduction conditions (n = 3, mean, SEM).

Article Snippet: Stimulated PBMCs were nucleofected with either in vitro -transcribed TALEN mRNA, or ribonucleoprotein (RNP) complexes of Cas9 (Alt-R ® S. p.HiFi Cas9 V3, IDT) or Cas12a (Alt-R ® AsCas12a Ultra, IDT) protein and their respective guides, according to the timeline ( ).

Techniques: Transduction

Journal: The CRISPR Journal

Article Title: Generation of Sex-Reversed Female Clonal Mice via CRISPR-Cas9-Mediated Y Chromosome Deletion in Male Embryonic Stem Cells

doi: 10.1089/crispr.2020.0074

Figure Lengend Snippet: Efficient elimination of the Y chromosome in mouse embryonic stem (ES) cells using CRISPR-Cas9. (A) Mouse Y chromosome and indicated relevant genes. (B) Scheme illustration of CRISPR-mediated Y chromosome elimination in mouse ES cells. (C) Y chromosome deletion rates as determined by Uba1y loss in targeted cells with varying concentrations of co-transfection green fluorescent protein (GFP) plasmid. BS: blinded selection.

Article Snippet: Duplex gRNAs were then incubated with recombinant Cas9 protein (IDT; HiFi Cas9 nuclease V3, cat. no. 1081060) at room temperature for 20 min to form gRNA-Cas9 ribonucleoproteins (RNPs), followed by co-delivery with a green fluorescent protein (GFP) plasmid (Addgene, Watertown, MA; cat. no. 42028).

Techniques: CRISPR, Cotransfection, Plasmid Preparation, Selection

Journal: bioRxiv

Article Title: Beyond polyA: scalable single-cell total RNA-seq unifies coding and non-coding transcriptomics

doi: 10.1101/2025.08.08.669394

Figure Lengend Snippet: a. Schematic overview of the TotalX protocol. Total RNA is polyadenylated and reverse-transcribed using a custom template-switching oligo (dUTSO). After reverse transcription, the TSO is digested with UDG and rRNA is depleted at the pre-amplified cDNA level using Cas9-based DASH. Long (>400 bp) and short (<400 bp) fragments are indexed separately, with optional inclusion of a gel-purified miRNA fraction (~18–50 bp) and pooled for sequencing. The schematic was designed using https://BioRender.com . b. Gene detection efficiency across technologies. Comparison of average number of genes per cell as a function of UMIs for TotalX (green), VASA-seq (orange), and 10xGenomics 3′ chemistry (blue), across binned depths. c. Unique genes detected per cell after UMI downsampling . Gene detection following normalization to 20,000 UMIs per cell. TotalX yields high gene complexity similar to VASA-seq and higher than standard 10xGenomics Chromium 3′ workflow. d. Total number of unique genes detected per RNA biotype. Radial plots show numbers of unique genes detected in a representative experiment for each method, broken down by RNA biotype: protein-coding, lncRNA, miscRNA, miRNA, snoRNA, snRNA, tRNA, and histone RNA. Ratios represent the proportion of detected genes relative to the total number of annotated genes within each biotype. Only genes detected in 10 or more cells were counted. e. Improved detection of miRNAs using mixed library input. Scatter plot shows average CPM per cell of TotalX alone (x-axis) vs. TotalX with added miRNA fraction [TotalX-miRNA(+)] (y-axis) in HEK293T cells. Known HEK293T-specific miRNAs (red) reach expression levels comparable to low/moderate protein-coding genes (blue). Inset: proportion of reads mapping to genome, indicating a tradeoff with miRNA inclusion. f. Coverage profiles for selected miRNAs. Read depth plots for MIR17 , MIR222 , and MIR221 showing mature miRNA arms (grey regions).

Article Snippet: Cas9 Nuclease V3 (NEB, 1081059), tracrRNA (IDT, 1072532), and a custom pool of 57 crRNAs (IDT) designed based on Smart-seq-total DASH oligos ( Supplementary Table 1 ).

Techniques: Reverse Transcription, Amplification, Purification, Sequencing, Comparison, Expressing

Journal: bioRxiv

Article Title: Beyond polyA: scalable single-cell total RNA-seq unifies coding and non-coding transcriptomics

doi: 10.1101/2025.08.08.669394

Figure Lengend Snippet: a. Schematic overview of the TotalX workflow. Total RNA is enzymatically polyadenylated and reverse transcribed using a custom template-switching oligo (TSO). The TSO is then selectively removed, followed by Cas9-based rRNA depletion (DASH) at the cDNA level. Resulting cDNA fragments are size-separated, with short (<400 bp) and long (>400 bp) fractions indexed independently and pooled for sequencing. An optional gel-purified miRNA fraction (~18–50 bp) enables targeted enrichment of mature small RNAs. b. Structure of the short-fragment indexed library. The short-fragment indexed libraries can optionally undergo Pippin gel selection system to obtain miRNA-enriched libraries. c. Workflow for combining short RNA, miRNA-enriched and standard cDNA fractions. The resulting composite library captures a broad range of transcript sizes and biotypes. d. Cell Ranger-based data analysis pipeline of TotalX. e. Examples of gene coverage profiles for representative miRNAs located within the lncRNA locus (top), intron of a protein coding gene (middle) and exon of a protein coding gene (bottom). Bam file used for the coverage plots was generated with the modified Cell Ranger pipeline.

Article Snippet: Cas9 Nuclease V3 (NEB, 1081059), tracrRNA (IDT, 1072532), and a custom pool of 57 crRNAs (IDT) designed based on Smart-seq-total DASH oligos ( Supplementary Table 1 ).

Techniques: Reverse Transcription, Sequencing, Purification, Selection, Generated, Modification