Journal: Veterinary World

Article Title: Synergistic enhancement of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 -mediated gene editing in porcine zygotes through combined lipofection and electroporation of cationic lipid-packaged ribonucleoproteins

doi: 10.14202/vetworld.2025.3806-3814

Figure Lengend Snippet: Schematic of the experimental design. Zona pellucida-free zygotes were edited with gRNA targeting either beta-1,4-N-acetyl-galactosaminyl transferase 2 ( B4GALNT2 ) or growth hormone receptor ( GHR ) genes by combining EP or transfection. IVM = In vitro maturation, IVF = In vitro fertilization, LP2000 = Lipofectamine 2000, RNP = Cas9-gRNA ribonucleoprotein complex, EP = Electroporation using RNPs, TL = Transfection through lipofectamine, EPL = Electroporation with lipofectamine-packaged RNPs.

Article Snippet: This buffer included 100 ng/μL Cas9 protein (Guide-it Recombinant Cas9, Takara Bio, Shiga, Japan; 632641) and 100 ng/μL gRNAs (Alt-R CRISPR crRNAs and tracrRNA; IDT) targeting B4GALNT2 (5′-ATGTGACGCCTTCGGG CATC-3′; 109047933) or GHR (5′-CTGTTGACCTTGGCAGTGGC-3′; 109047932).

Techniques: Transfection, In Vitro, Electroporation

Journal: bioRxiv

Article Title: RNA splicing dynamics in CD8 T cells uncovers isoforms that impact T cell-mediated cancer immunotherapy

doi: 10.1101/2025.09.07.674706

Figure Lengend Snippet: a, SpliceSeek CRISPR editing strategy. Cas9 inserts indels at either the 3′ splice acceptor site (3′SS; left) or the 5′ splice donor site (5′SS; right), disrupting U1/U2AF recognition and triggering exon skipping, alternative splice-site usage or intron retention, without altering the coding sequence. Created in BioRender. b , Schematic of antibody epitopes: V-domain antibody (purple) binds only the canonical SLAMF6 isoform, whereas C-domain antibody (blue) recognizes all isoforms. Created in BioRender. c , Representative flow-cytometry histograms of Jurkat T cells edited with control or 3′SS guides and stained pan–SLAMF6 C-domain antibody (right) or a V-domain antibody specific to the canonical isoform (left). d , Quantification of the percentage of SLAMF6 V-domain and C-domain positive cells (mean ± SD; n = 3). e–g , Optimization of Cas9 electroporation parameters in primary human T cells transduced with a SLAMF6 knockout guide, assayed by SLAMF6 surface staining. e , Representative flow-cytometry histograms showing SLAMF6 expression after electroporation using different Lonza 2b programs. f , Percentage of SLAMF6⁺ cells for each electroporation program (mean ± SD, n = 3 donors). g , Representative histograms of SLAMF6 expression following T-007 electroporation with increasing Cas9 protein doses. h , Final editing efficiency under the optimized conditions (T-007 program + 5 µg Cas9): representative FACS density plots of SLAMF6 versus NY-ESO-1 TCR expression, with percentages of SLAMF6-negative cells in the SLAMF6 knockout guide condition. i-l , Flow cytometry of primary human T cells 6 hours after either no stimulation (left) or re-activation (right). i, k , Representative intracellular FACS dot plots of IFN-γ versus CD107a (i) or granzyme B (k), with quadrant percentages. j , Quantification of IFN-γ⁺CD107a⁺ cells (mean ± SD; n = 3). l , Quantification of IFN-γ⁺GZMb⁺ cells (mean ± SD; n = 4) P values were determined by two-way ANOVA with Bonferroni correction (d), Kruskal–Wallis test with Dunnett’s

Article Snippet: Twenty-four hours later, cells were electroporated with 5 μg recombinant Cas9 protein using the Amaxa human T cell Nucleofection kit (Lonza, VPA-1002, program T-007).

Techniques: CRISPR, Sequencing, Flow Cytometry, Control, Staining, Electroporation, Transduction, Knock-Out, Expressing, Activation Assay

Journal: bioRxiv

Article Title: RNA splicing dynamics in CD8 T cells uncovers isoforms that impact T cell-mediated cancer immunotherapy

doi: 10.1101/2025.09.07.674706

Figure Lengend Snippet: a , Schematic of CRISPR-Cas9 targeting of SLAMF6 splice sites. Disruption of the 3′ splice acceptor (3′SS; top) permits use of alternative downstream acceptor sites and exon skipping, whereas disruption of the 5′ splice donor (5′SS; bottom) yields exon skipping only. Created in BioRender . b–c , SLAMF6 isoform expression following splice-site editing in Jurkat T cells transduced with control, 3′SS or 5′SS guides. b , RT-PCR of SLAMF6 isoform and house-keeping control (GAPDH). c , RT-qPCR quantification of each isoform (mean ± SD; Ctrl n = 5, 3′SS n = 5, 5′SS n = 2). d , Schematic of the pooled SpliceSeek screen in primary, antigen-specific, human T cells. Created in BioRender. e–f, Reference gene knockout screen in Jurkat T cells activated by co-culture with T2 peptide loaded cell (n = 3 independent experiments). e, Volcano plot of sgRNA enrichment; red - guides enriched in IL-2-high cells; blue - guides enriched in IL-2-low cells. f, Density strips of guide-level log₂ FC for selected targets (top panel shows overall guide distribution). g-h, Reference gene knockout screen in primary human T cells (n=3 donors). g, Volcano plot of sgRNA enrichment; red - guides enriched in IFN-γ-high cells; blue - guides enriched in IFN-γ-low cells. h, Density strips of guide-level log₂ FC for selected targets (top panel shows overall guide distribution) i, Intracellular IL-2 production in Jurkat cells following overnight activation with PMA and ionomycin, comparing BSG knockout and control guides. j, Intracellular IFN-γ production in primary PBMCs after overnight co-culture with T2 cells, comparing BSG knockout and control guides. Data in b is representative of three independent experiments. P values were determined by mixed-effects model with Dunnett’s correction ( c ), and two-way ANOVA ( i,j ). *P < 0.05 **P < 0.01, ***P < 0.001.

Article Snippet: Twenty-four hours later, cells were electroporated with 5 μg recombinant Cas9 protein using the Amaxa human T cell Nucleofection kit (Lonza, VPA-1002, program T-007).

Techniques: CRISPR, Disruption, Expressing, Transduction, Control, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Gene Knockout, Co-Culture Assay, Activation Assay, Knock-Out

Journal: bioRxiv

Article Title: RNA splicing dynamics in CD8 T cells uncovers isoforms that impact T cell-mediated cancer immunotherapy

doi: 10.1101/2025.09.07.674706

Figure Lengend Snippet: a–b , Reference CRISPR–Cas9 knockout screen in Jurkat T cells (n = 3 independent experiments) stimulated with PMA/ionomycin and sorted into IL-2–high and IL-2–low fractions. a, Volcano plot of sgRNA enrichment; red - guides enriched in IL-2-high cells; blue - guides enriched in IL-2-low cells. b, Guide-level log₂ FC density strips for selected targets (top strip shows the overall guide distribution). c-d , Pearson correlation of sgRNA enrichment (log₂ FC values) in Jurkat cells activated with PMA/ionomycin versus peptide-loaded T2 cells (c) and primary PBMCs versus Jurkat PMA/ionomycin activation (d). Shaded band indicates the 95% confidence interval. e-h BSG (CD147) knockout efficiency validation in jurkats cells (e-f, mean ± SD; 2 different knockout guides, 3 independent experiments per guide) and PBMCs (g-h, mean ± SD; n = 3 donors). e and g are representative histograms of flow cytometry. f and h, are Quantification of BSG⁺ cells. i-j, Representative flow cytometry of cytokine expression in BSG KO cells vs controls in jurkat cells (i) and PBMCs (j). P values were determined by two-tailed paired t-test (f,h). ***P < 0.001.

Article Snippet: Twenty-four hours later, cells were electroporated with 5 μg recombinant Cas9 protein using the Amaxa human T cell Nucleofection kit (Lonza, VPA-1002, program T-007).

Techniques: CRISPR, Knock-Out, Stripping Membranes, Activation Assay, Biomarker Discovery, Flow Cytometry, Expressing, Two Tailed Test