synthetic modified sgrnas  (Synthego Inc)

Journal: bioRxiv

Article Title: An InDel Genomic Variant within a Bifunctional Super-Enhancer for LINC00636 and CD47 Regulation in Breast Cancer

doi: 10.1101/2025.11.05.684493

Figure Lengend Snippet: LINC00636 RNA expression is regulated by a bifunctional SE. (A) LINC00636 and CD47 RNA expression levels decrease upon BET inhibition in BCCL, and this reduction occurs at a greater extent in the cells where the SE is present. Cells were treated with 1 μM JQ1 or I-BET151 for 6 hours and mRNA levels were analyzed by qPCR. DMSO was used as vehicle control. Two-way ANOVA was performed, *p<0.05, **p<0.01, ***p<0.001. (B) CRISPRa of the SE locus increases LINC00636 and CD47 RNA expression levels, and this activation occurs at a greater extent in the cells where the SE is present. dCas9-VP64-expressing cells were transfected with scramble sgRNA or sgRNA targeting the E5 core element within the SE. mRNA levels were analyzed by qPCR 48 hours after transfection. Two-way ANOVA was performed, *p<0.05, **p<0.01, ***p<0.001. (C) LINC00636 overexpression does not affect CD47 RNA expression levels. Left image shows the CRISPRa approach used for LINC00636 overexpression targeting LINC00636 TSS. The plots show LINC00636 and CD47 RNA levels in dCas9-VP64-expressing MCF7 cells transfected with sgRNAs targeting two different sites on LINC00636 TSS. mRNA levels were analyzed by qPCR 48 hours after transfection. ANOVA was performed, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Synthetic modified sgRNAs (Synthego) were used for deleting the 8bp insertion within the bifunctional SE.

Techniques: RNA Expression, Inhibition, Control, Activation Assay, Expressing, Transfection, Over Expression

Journal: bioRxiv

Article Title: An InDel Genomic Variant within a Bifunctional Super-Enhancer for LINC00636 and CD47 Regulation in Breast Cancer

doi: 10.1101/2025.11.05.684493

Figure Lengend Snippet: Deletion of the insertion enhances resistance to cellular stress and reduces infiltration of pro-inflammatory macrophages. (A) MCF7Δ#1 and MCF7Δ#2 clones maintain spheroid integrity in 3D cultures, while MCF7 cells lose compact spherical morphology. MCF7Δ#1 and MCF7Δ#2 clones or MCF7 control cells were seeded on plates pre-coated with Matrigel and imaged at day 7. Three representative images per condition are shown. Scale bars represent 100 μm. (B) Deleting the insertion reduces cell death in 3D cultures. MCF7Δ#1 and MCF7Δ#2 clones or MCF7 control cells were seeded on plates pre-coated with Matrigel and imaged at day 7 upon staining with BD Via-Probe™ Green Nucleic Acid Stain. Panels show bright field, fluorescence (cell death) and merged images. Scale bars represent 100 μm. Number of dead cells per spheroid was quantified using ImageJ. (C) CRISPRa of the bifunctional SE in MCF7 cells restores spheroid integrity in 3D cultures. MCF7 cells expressing dCas9-VP64 and sgRNAs scramble control or targeting the E5 core element within the SE were seeded on plates pre-coated with Matrigel and imaged on day 7. Top image shows the CRISPRa strategy used to activate the bifunctional SE. Scale bars represent 100 μm. (D) Deleting the insertion increases resistance to apoptosis induced by glucose deprivation in MCF7. MCF7Δ#1 and MCF7Δ#2 clones or MCF7 control cells were grown in media without glucose for 48 hours and the percentage of apoptotic cells was quantified by Annexin V staining and flow cytometry. Two-way ANOVA, **p<0.01, ***p<0.001. (E) CD47 overexpression increases resistance to apoptosis induced by glucose deprivation in MCF7. MCF7 cells expressing dCas9-VP64 and sgRNAs scramble control or sRNAs targeting LINC00636 or CD47 promoters were grown in media without glucose for 48 hours and the percentage of apoptotic cells was quantified by Annexin V staining and flow cytometry. Top image shows the CRISPRa strategy used for LINC00636 or CD47 overexpression. Two-way ANOVA, *p<0.05. (F) LINC00636 overexpression increases senescence in breast cancer cells. MCF7, T47D or HCC1954 cells expressing dCas9-VP64 and sgRNAs scramble control or sgRNAs targeting LINC00636 TSS were studied. 48 hours post-seeding, senescence was analyzed by b-galactosidase staining, images were captured, and the percentage of senescent (blue) cells was quantified. Left panel shows representative images of MCF7 cells overexpressing LINC00636 or control. Scale bars represent 110 μm. Two-way ANOVA, ***p<0.001. (G) LINC00636 overexpression increases expression of senescence markers in MCF7. MCF7 cells expressing dCas9-VP64 and sgRNAs scramble control or sgRNAs targeting LINC00636 TSS were studied. 48 hours post-seeding, senescence markers IL1A, IL1B, IL6 and IL8 were analyzed by qPCR. Two-way ANOVA, ***p<0.001. (H) Infiltration of CD80+ macrophages decreases in tumors derived from insertion-deleted MCF7Δ#1 and MCF7Δ#2 clones compared to parental MCF7 cells. CD80 expression was analyzed by flow cytometry upon collection of the breast tumors. Left graph shows CD80+ cells infiltrating the tumors as percentage of macrophages. Right graph is the histogram of CD80-FITC fluorescence in CD80+ macrophages. ANOVA, *p<0.05, ***p<0.001.

Article Snippet: Synthetic modified sgRNAs (Synthego) were used for deleting the 8bp insertion within the bifunctional SE.

Techniques: Clone Assay, Control, Staining, Fluorescence, Expressing, Flow Cytometry, Over Expression, Derivative Assay

Journal: bioRxiv

Article Title: Base-editing a single missense mutation in A20 enhances CAR-T cell efficacy

doi: 10.1101/2025.10.05.680562

Figure Lengend Snippet: (A) Schematic illustration of the human TCR-T cell repetitive stimulation assay. (B) Normalized cancer growth curves of NY-ESO-1 + A375 melanoma target cells co-cultured with 1G4 TCR-T cells. Right panels depict co-cultures with TCR-T cells that had already undergone either 2, 4 or 6 rounds of repetitive tumor cell stimulation (rep stim; 14, 19 and 23 days in culture, respectively). Left panels depict co-cultures with control TCR-T cells that were matched for time in culture but had not undergone repetitive tumor cell stimulation (no rep stim). Effector:target (E:T) ratio = 1:1. Growth curves depict mean ± SEM of n = 3 technical replicates from n = 2 T cell donors. (C) (Top row) Normalized supernatant concentrations (pg/ml) of secreted effector molecules, as determined by LEGENDplex. Analysis shows TCR-T cells that had undergone either 1, 4, or 6 rounds of repetitive stimulation (10, 19 or 23 days in culture, respectively). (Bottom row) Normalized IR expression on TCR-T cells. Analysis shows TCR-T cells after 2 or 6 rounds of repetitive tumor antigenic stimulation (rep stim) compared with TCR-T cells matched for time in culture but not subjected to repetitive stimulation (control). MFI = median fluorescence intensity. Each technical replicate was normalized to the mean value of the control (no rep stim; bottom row) or 10 days in culture group (top row) for the corresponding donor. Bar graphs depict mean ± SEM of n = 2 donors, with n = 2-3 technical replicates per group per donor. Significance was assessed using paired t test. *p<0.05; **p<0.01. (D) Donor to donor correlation of TF versus T0. Highlighted genes include A20/ TNFAIP3 (red) and genes with known roles in T cell fitness (black). (E) Volcano plot comparing gene level scores from TFctrl versus T0 groups (left; n = 2 T cell donors) or from TI versus T0 groups (right; n = 1 T cell donor). Highlighted genes include A20/ TNFAIP3 (red) and genes with known roles in T cell fitness (black). (F) Normalized dividing TCR-T cells (CellTrace Violet lo ), as determined by proliferation assay after 72 h of stimulation. Each technical replicate was normalized to mean value of AAVS1 for corresponding donor. Bar graphs depict mean ± SEM of n = 3 donors with n = 3 technical replicates per group per donor. Significance was assessed by paired t test. (G) Normalized cancer growth curves of NY-ESO-1 + A375 melanoma target cells. TCR-T cells after 5 rounds of repetitive stimulation were co-cultured with A375 cells at E:T ratio of 1:4. Growth curves depict mean ± SEM of n = 3 technical replicates from n = 1 T cell donor. (H) Individual NALM6 growth curves of mice treated with CAR-T cells. Left panel depicts different donor than that used in . Right panel depicts same donor as used in , but different sgRNA used to target A20 KO . n = 6 mice per group. CR = complete response. (I) Survival analysis for (H). Significance was assessed using a log-rank test. (J) Absolute numbers of total (CD4 + and CD8 + combined) CAR-T cells isolated from bone marrow of NALM6-bearing mice 14 days after CAR-T cell injection (mean ± SEM, n = 2 T cell donors, n = 3 mice per group per donor). Significance was assessed using unpaired t test with Welch’s correction. (K) Differentiation status of CD4 + or CD8 + CAR-T cells from (e). Naive = CD45RA + CD62L + ; central memory (Tcm) = CD45RA − CD62L + ; effector memory (Tem) = CD45RA − CD62L − ; effector (Teff) = CD45RA + CD62L − (mean ± SEM, n = 2 T cell donors, n = 3 mice per group per donor). (D,E) Statistics were determined by MAgECK analysis.

Article Snippet: Next, 2 μg of CBE mRNA along with 60 nmols of synthetic modified sgRNA (Synthego) targeting either B2Mi, A20 KO or A20 ZF7 (sgRNAs specified in Supp.

Techniques: Cell Culture, Cell Stimulation, Control, Expressing, Fluorescence, Proliferation Assay, Isolation, Injection

Journal: bioRxiv

Article Title: Base-editing a single missense mutation in A20 enhances CAR-T cell efficacy

doi: 10.1101/2025.10.05.680562

Figure Lengend Snippet: (A) Immunoblot of A20 expression by Cas9- or base-edited (CBE) human T cells that were either not activated, or activated with anti-CD3/CD28 for 24 hours. n = 1 donor. (B) Individual NALM6 growth curves of mice treated with Cas9-edited CAR-T cells or with TRAC KO (no CAR) T cells (n = 1 T cell donor different from = 6-7 mice per group). CR = complete response. Leukemia burden was quantified via BLI. Radiance units = photons/s/cm 2 /steradian. (C) Individual NALM6 growth curves of mice that controlled initial challenge and underwent iterative rechallenge (black arrows). (D) CBE screen (top) and ESM1b heatmap (bottom) from , zoomed in on A20 ZF7 motif. sgRNA target (Cys779) highlighted in green. (E) Representative dot plots depicting β2m expression on B2Mi CAR-T cells 36 hours after base-editing. Inset = percentage of β2m + cells. (F) Normalized dividing cells (CellTrace Violet lo ), as determined via proliferation assay of CAR-T cells after 72 h of stimulation. (G) Normalized growth curves of A375-CD19 cells. CAR-T cells either without prior repetitive stimulation (no rep stim) or after 3 rounds of repetitive stimulation (rep stim) were co-cultured with A375-CD19 cells at an E:T ratio of 1:1 (mean ± SEM of n = 3 technical replicates; n = 2 donors). (H) Normalized expression of activation markers by CD4 + (top) and CD8 + (bottom) CAR-T cells before (pre) and after (post) 3 rounds of tumor stimulation. (I) Fold change of base-edited CD19 CAR-T cells cultured without cytokine either in the absence (left) or presence (right) of A375-CD19 target cells (E:T ratio 1:1). (J) Normalized growth curves of CD19-negative A375 melanoma cells co-cultured with CD19 CAR-T cells (E:T 1:1), or cultured without CAR-T cells. (F,H) Bar graphs depict mean ± SEM of n = 2 donors with n = 3 technical replicates per donor. Each technical replicate was normalized to mean of B2Mi group for corresponding donor and condition. Significance was assessed using paired t test. (I,J) All curves show mean ± SEM of n = 3 technical replicates from n = 2 T cell donors. *p<0.05.

Article Snippet: Next, 2 μg of CBE mRNA along with 60 nmols of synthetic modified sgRNA (Synthego) targeting either B2Mi, A20 KO or A20 ZF7 (sgRNAs specified in Supp.

Techniques: Western Blot, Expressing, Proliferation Assay, Cell Culture, Activation Assay

Journal: bioRxiv

Article Title: Base-editing a single missense mutation in A20 enhances CAR-T cell efficacy

doi: 10.1101/2025.10.05.680562

Figure Lengend Snippet: (A) Individual NALM6 growth curves of mice treated with Cas9-edited CAR-T cells or with TRAC KO (no CAR) T cells (n = 1 T cell donor, n = 5-10 mice per group). CR = complete response. Leukemia burden was quantified via BLI. Radiance units = photons/s/cm 2 /steradian. (B) Survival analysis corresponding to (A). Significance was assessed using log-rank test. (C) Schematic illustration for the generation of base-edited CD19 TRAC -CAR-T (top) or 1G4 TCR-T (bottom) cells. Cas12a RNP carrying TRAC -targeting sgRNAs (CAR-T only), cytosine base-editor (CBE)-encoding mRNA, and a CBE sgRNA were introduced into T cells via electroporation. (D) Normalized supernatant concentrations (pg/ml) of secreted effector molecules from base-edited CD19 CAR-T cells, as determined by LEGENDplex. CAR-T cells were analyzed before (pre) or after (post) 3 rounds of repetitive stimulation. (E) Normalized IR expression by CD4 + and CD8 + CD19 CAR-T cells either before (pre) or after (post) 5 rounds of repetitive stimulation. (F) Schematic illustration for the generation of NF-κB activity reporter CAR-T or TCR-T cells. Cells were electroporated with Cas12a RNP carrying B2Mi-targeting sgRNAs, then transduced with AAV carrying NF-κB reporter construct. For base-edited cells, steps in (C) were concurrently performed. For Cas9-edited cells, Cas9 RNPs with sgRNAs targeting AAVS1, A20 KO or A20 ZF7 were electroporated in lieu of CBE mRNA and associated sgRNAs. (G) Normalized NF-kB-CFP MFI of Cas9 or base-edited CD19 CAR-T (left) or 1G4 TCR-T (right) cells before (unstim) or after 12 h stimulation (stim) with antigen-expressing tumor cells (E:T 1:2). (D,E,G) Bar graphs depict mean ± SEM of n = 2 donors with n = 3 technical replicates per donor. Each technical replicate was normalized to mean of control (AAVS1 or B2Mi) group for corresponding donor and condition. Significance was assessed using paired t test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: Next, 2 μg of CBE mRNA along with 60 nmols of synthetic modified sgRNA (Synthego) targeting either B2Mi, A20 KO or A20 ZF7 (sgRNAs specified in Supp.

Techniques: Electroporation, Expressing, Activity Assay, Transduction, Construct, Control