t7 endonuclease  (New England Biolabs)

Journal: Nature Communications

Article Title: CD73 disruption with tumor-specific CRISPR/Cas9 nanoparticles reduces adenosine-mediated immunosuppression and amplifies response to chemotherapy

doi: 10.1038/s41467-026-77547-4

Figure Lengend Snippet: a Size distribution of iRGD-DC@CaP as determined by dynamic light scattering, and morphology of iRGD-DC@CaP as revealed by TEM images. Scale bar: 200 nm. b , c DOX and Cas9 release from iRGD-DC@CaP under different situations ( n = 3 biologically independent samples). Representative flow cytometry analysis images ( d ) and quantitative analysis ( e ) of cellular uptake in B16F10 cells following a 4 h incubation with iRGD-DC@CaP or controls ( n = 3 biologically independent samples). f Confocal laser scanning microscopy images of B16F10 cells following a 4 h incubation with iRGD-DC@CaP or controls. Cas9 was labeled with Alexa Fluor 647 (AF647; red), and the nuclei were stained with DAPI (blue). Scale bars: 50 µm. T7EI assay ( g ) and quantitative analysis ( h ) of indel frequencies of the Nt5e gene in B16F10 cells transfected with iRGD-DC CD73 @CaP or controls for 48 h ( n = 3 biologically independent samples). i Mutation rates of Nt5e examined by NGS analysis in B16F10 cells treated with iRGD-DC CD73 @CaP. j , k Western blot ( j ) and flow cytometry quantitative analysis ( k ) of CD73 protein expression in B16F10 cells after 72 h incubation with iRGD-DC CD73 @CaP or controls ( n = 3 biologically independent samples). l The ADO contents in the cell medium of B16F10 cells after 72 h incubation with iRGD-DC CD73 @CaP or controls detected by ELISA kits ( n = 3 biologically independent samples). The experiments for f and i were independently repeated three times with similar results. Data is presented as the mean ± SD. Statistical difference was assessed in ( e , h , j , k and l ) using one-way ANOVA with Dunnett’s multiple comparison test by comparing the iRGD-DC CD73 @CaP group with each of the other treatment groups. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. G1: saline (negative control); G2: Free Cas9 RNP; G3: iRGD-modified nanoparticles loaded with scrambled sgRNA (iRGD-DC Scr @CaP, negative control); G4: Lipofectamine™ CRISPRMAX™-mediated delivery of Cas9/sgCD73 (MAX Cas9/sgCD73, positive control); G5: nanoparticle without iRGD loaded with Cas9/sgCD73 (DC CD73 @CaP); G6: iRGD-modified nanoparticle loaded with Cas9/sgCD73 (iRGD-DC CD73 @CaP). The exact P value and Source data are provided as a Source Data file.

Article Snippet: T7 endonuclease I (T7EI) enzyme, EnGen Cas9 NLS S. pyogenes, HiScribe T7 High Yield RNA Synthesis Kit were purchased from New England BioLabs (NEB).

Techniques: Dynamic Light Scattering Assay, Morphology, Flow Cytometry, Analysis, Incubation, Confocal, Laser-Scanning Microscopy, Labeling, Staining, T7EI Assay, Transfection, Mutagenesis, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Comparison, Saline, Negative Control, Modification, Positive Control

Journal: Nature Communications

Article Title: CD73 disruption with tumor-specific CRISPR/Cas9 nanoparticles reduces adenosine-mediated immunosuppression and amplifies response to chemotherapy

doi: 10.1038/s41467-026-77547-4

Figure Lengend Snippet: a Fluorescence imaging of B16F10 tumor-bearing C57BL/6 mice following injection of iRGD-DC@CaP and controls ( n = 3 mice). b Quantitative analysis of AF647-Cas9 fluorescence from major organs in mice bearing B16F10 at 1 h after intravenous injection of iRGD-DC@CaP or controls ( n = 3 mice). “% ID/g” represents the percentage of the injected dose per gram of tissue. The exact P value: DC@CaP vs. iRGD-DC@CaP ** P = 0.0087. c Schematic illustration of B16F10 tumor therapy in vivo. Created in BioRender. Sun, X. (2026) https://BioRender.com/f4h4ymp . d Individual and average tumor growth curves of B16F10 following different treatments ( n = 5 mice). e Tumor weight and the tumor inhibition ratio following different treatments ( n = 5 mice). f Survival analysis of the mice following different treatments ( n = 6 mice). P values were determined by the log-rank test. g Indel frequency of Nt5e loci measured by T7EI assay in B16F10 tumor tissues with the indicated treatments. The experiment was independently repeated three times with similar results. h Western blotting and relative quantitative analysis of CD73 proteins in B16F10 tumor tissues with the indicated treatments ( n = 3 biologically independent samples). i The flow cytometry quantitative analysis of the CD73 expression on tumor cells (CD45 − ), endothelial cells (CD45 − CD31 + ), cancer-associated fibroblasts (CAFs) (CD45 − CD31 − GP38 + ), macrophages (CD45 + CD11b + F4/80 + ), DCs (CD45 + CD11c + MHC-II + ), Tregs (CD45 + CD4 + CD25 + Foxp3 + ) and MDSCs (CD45 + CD11b + Gr-1 + ) in B16F10 tumor tissues after indicated treatments ( n = 5 mice). j The ADO contents in the tumor after different treatments ( n = 5 mice). Data is presented as the mean ± SD. Statistical differences were assessed using one-way ANOVA in ( b , e , h , i and j ) or two-way ANOVA in ( d ) with Dunnett’s multiple comparison test by comparing the iRGD-DC CD73 @CaP group with each of the other treatment groups. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. G1: saline (negative control); G2: iRGD-modified nanoparticles loaded with scrambled sgRNA (iRGD-DC Scr @CaP, negative control); G3: iRGD-modified nanoparticle loaded with Cas9/sgCD73 (iRGD-DC CD73 @CaP). The exact P value and Source data are provided as a Source Data file.

Article Snippet: T7 endonuclease I (T7EI) enzyme, EnGen Cas9 NLS S. pyogenes, HiScribe T7 High Yield RNA Synthesis Kit were purchased from New England BioLabs (NEB).

Techniques: Fluorescence, Imaging, Injection, Analysis, Mouse Assay, Tissue, In Vivo, Inhibition, T7EI Assay, Western Blot, Flow Cytometry, Expressing, Comparison, Saline, Negative Control, Modification

Journal: Nature Communications

Article Title: CD73 disruption with tumor-specific CRISPR/Cas9 nanoparticles reduces adenosine-mediated immunosuppression and amplifies response to chemotherapy

doi: 10.1038/s41467-026-77547-4

Figure Lengend Snippet: a Schematic illustration of experimental design for 4T1 tumor therapy in vivo. b , c Average ( b ) and individual ( c ) tumor growth curves of 4T1 following different treatments ( n = 5 mice). d Tumor weight and tumor inhibition ratio following different treatments ( n = 5 mice). e Survival analysis of the mice following different treatments ( n = 6 mice). f Indel frequency of Nt5e loci measured by T7EI assay in 4T1 tumor tissues with the indicated treatments. The experiment was independently repeated three times with similar results. g Western blot and relative quantitative analysis of CD73 protein expression in 4T1 tumor tissues with the indicated treatments ( n = 3 biologically independent samples). h The ADO contents in the tumor after different treatments ( n = 5 mice). i–t Flow cytometry quantitative analysis of CD80 + CD86 + DCs ( i ), absolute number of CD8 + T cells ( j ), CD3 + CD8 + T cells ( k ), CD4 + CD25 + Foxp3 + Tregs ( l ), CD8 + T/Tregs ratio ( m ), CD3 − CD49b + NK cells ( n ), F4/80 + CD86 + cells ( o ), F4/80 + iNOS + cells ( p ), F4/80 + CD206 + cells ( q ), F4/80 + Arg1 + cells ( r ), F4/80 + CD86 + M1/F4/80 + CD206 + M2 ratio ( s ), and CD11b + Gr-1 + MDSCs ( t ) in tumor after different treatments ( n = 5 mice). u–w Contents of the IL-2 ( u ), TNF-α ( v ), and IFN-γ ( w ) in tumor tissues after different treatments ( n = 5 mice). Data is presented as the mean ± SD. Statistical differences were analyzed by two-way ANOVA with Dunnett’s multiple comparison test in ( b ), one-way ANOVA with Dunnett’s multiple comparison test in ( d , g , and h–w ) by comparing the iRGD-DC CD73 @CaP group with each of the other treatment groups. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. G1: saline (negative control); G2: iRGD-modified nanoparticles loaded with scrambled sgRNA (iRGD-DC Scr @CaP, negative control); G3: iRGD-modified nanoparticle loaded with Cas9/sgCD73 (iRGD-DC CD73 @CaP). The exact P value and Source data are provided as a Source Data file.

Article Snippet: T7 endonuclease I (T7EI) enzyme, EnGen Cas9 NLS S. pyogenes, HiScribe T7 High Yield RNA Synthesis Kit were purchased from New England BioLabs (NEB).

Techniques: In Vivo, Inhibition, Analysis, T7EI Assay, Western Blot, Expressing, Flow Cytometry, Comparison, Saline, Negative Control, Modification