grna expression plasmid  (Addgene inc)

Journal: Cell Death & Disease

Article Title: Chondroitin sulfate shapes the unfolded protein response in osteosarcoma to sustain cancer cell survival during ER stress

doi: 10.1038/s41419-026-09151-9

Figure Lengend Snippet: List of CRISPR gRNA design and PCR primers.

Article Snippet: Briefly, 3 gRNA sequences per gene were designed for B4GALT7, CHST11 , and CHST12 genes using the online predictor tool ( https://horizondiscovery.com/en/ordering-and-calculation-tools/crispr-design-tool ) and were inserted into the gRNA expression plasmid (Addgene plasmid, #68370).

Techniques: CRISPR

Journal: bioRxiv

Article Title: SMARCAL1 is a candidate therapeutic target for ALT-positive tumors

doi: 10.64898/2026.02.15.704070

Figure Lengend Snippet: Effects of SMARCAL1 and/or FANCM loss in ALT-positive cancer cells (A-F) Detection by western blotting of SMARCAL1, phosho-RPA32 (S4/S8), γH2AX and FANCM levels in whole cell extracts of the specified cells treated as indicated. Vinculin, tubulin and H2AX are shown as a loading control. (G-H) Quantification of the fitness of OS384 (G) and SAOS2 (H) cells expressing Cas12a and transduced with the indicated gRNAs relative to the control gRNA. Columns represent the mean ± SEM of independent biological replicates (dots). The expected effect induced by dual gene targeting is indicated by the dashed red line. Statistical significance was determined using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. (I-J) Detection by RT-qPCR of the fold change in SMARCAL1 and FANCM mRNA levels in SAOS2 cells treated with the indicated siRNAs (I) or expressing Cas12a and the indicated gRNAs (J) relative to the control treatment (CTR). SMARCAL1 and FANCM mRNA levels in both cell lines were normalized to TBP mRNA levels. Significance levels are denoted as follows: *p < 0.05, ****p < 0.0001.

Article Snippet: In4mer arrays were ordered as individual eBlocks (IDT) and cloned into the lentiviral Cas12a gRNA expression backbone pRDA_052 (Addgene #136474) using the restriction cut site Esp3I.

Techniques: Western Blot, Control, Expressing, Transduction, Quantitative RT-PCR

Journal: bioRxiv

Article Title: SMARCAL1 is a candidate therapeutic target for ALT-positive tumors

doi: 10.64898/2026.02.15.704070

Figure Lengend Snippet: PRIMPOL-dependent cellular effects induced in ALT-positive cancer cells following SMARCAL1 loss (A-B) Quantification of the fitness of G292 cells transfected with the indicated cDNAs and siRNAs relative to the control condition (CTR siRNA or untreated). Columns represent the mean ± SEM of independent biological replicates (dots). Statistical significance was determined using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. (C) Detection by western blotting of the indicated proteins in whole cell extracts of G292 cells transfected with the indicated siRNAs. RPA32 is shown as a loading control. (D) Quantification of the number of ssTelo foci per nucleus in G292 cells transduced with the indicated siRNAs. Data are represented as box-and-whisker plots, showing the median (line), mean (+), 1 st to 3 rd quartiles (colored box), whiskers extending to the 10 th and 90 th percentiles, and individual outlier points. Data were obtained from 3 independent experiments. Statistical analysis was conducted as in (A-B). (E) Quantification of the fitness of SAOS2 cells expressing Cas12a and transduced with the indicated gRNAs, relative to the control conditions (CTR gRNA), as determined by cell counting. Columns represent the mean ± SEM of independent biological replicates (dots). Statistical analyses were conducted as in (D). Significance levels are denoted as follows: ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: In4mer arrays were ordered as individual eBlocks (IDT) and cloned into the lentiviral Cas12a gRNA expression backbone pRDA_052 (Addgene #136474) using the restriction cut site Esp3I.

Techniques: Transfection, Control, Western Blot, Transduction, Whisker Assay, Expressing, Cell Counting

Journal: bioRxiv

Article Title: SMARCAL1 is a candidate therapeutic target for ALT-positive tumors

doi: 10.64898/2026.02.15.704070

Figure Lengend Snippet: Telomeric foci and cell fitness in SMARCAL1-deficient ALT-positive cancer cells upon BLM or PRIMPOL loss (A-F) Quantification of the indicated colocalizing foci per nucleus in G292 (A-C) and SAOS2 (D-F) cells expressing Cas12a and transduced with the indicated gRNAs. Data are represented as box-and-whisker plots, showing the median (line), mean (+), 1 st to 3 rd quartiles (colored box), whiskers extending to the 10 th and 90 th percentiles, and individual outlier points. Data were obtained from 3 independent experiments. Statistical significance was determined using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. (G) Detection by western blotting of the indicated proteins in whole cell extracts of G292-Cas12a cells treated with the indicated gRNAs. Tubulin is shown as a loading control. (H) Quantification of the fitness of G292 cells expressing Cas12a and transduced with the indicated gRNAs, relative to the control conditions (CTR gRNA), as determined by cell counting. Graphical representation and statistical analysis were conducted as in (A-F). Columns represent the mean ± SEM of independent biological replicates (dots). Significance levels are denoted as follows: ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: In4mer arrays were ordered as individual eBlocks (IDT) and cloned into the lentiviral Cas12a gRNA expression backbone pRDA_052 (Addgene #136474) using the restriction cut site Esp3I.

Techniques: Expressing, Transduction, Whisker Assay, Western Blot, Control, Cell Counting

Journal: bioRxiv

Article Title: SMARCAL1 is a candidate therapeutic target for ALT-positive tumors

doi: 10.64898/2026.02.15.704070

Figure Lengend Snippet: Senescence-associated phenotypes in SMARCAL1-deficient cells and schematic representation of the interplay between BLM, SMARCAL1 and PRIMPOL in ALT-positive cancer cells (A) Representative images of nuclei and β-galactosidase fluorescence in OS384 cells transfected with the indicated siRNAs (left panel). Quantification of the percentage of senescent (β-gal)-positive cells following transfection with the indicated siRNAs (right panel). Columns represent the mean ± SEM of independent biological replicates (dots). Statistical significance was determined using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. (B-C) Quantification of IL6 mRNA levels by RT-qPCR in G292iDAXX (B) and SAOS2 cells expressing Cas12a (C) treated with the indicated siRNAs and gRNAs, respectively, with or without doxycycline (doxy) treatment to express DAXX, as indicated. IL6 mRNA levels were normalized to TBP mRNA levels and shown as fold change relative to the control condition (CTR). Data representation and statistical analysis were conducted as in (A). (D) Quantification of the fitness of SAOS2 cells transduced with the indicated gRNAs, with or without navitoclax (0.3 mM) treatment, relative to the control condition (CTR gRNA). Columns represent the mean ± SEM of independent biological replicates (dots). Statistical significance was determined using ordinary two-way ANOVA followed by Tukey’s multiple comparisons test. (E) Survival of G292iDAXX cells following treatment with A1331852 at the indicated concentrations. Cell survival is expressed as a fraction relative to the untreated control, and data represent the mean ± SEM of at least three replicates per condition. Statistical analysis was conducted as in (A). (F) Quantification of the fitness of G292 cells expressing Cas12a and transduced with the indicated gRNAs, with navitoclax (150 nM) treatment relative to the untreated condition. Graphical representation and statistical analysis were conducted as in (A). Significance levels are denoted as follows: ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (G) Graphical schematic illustrating the functional interplay between BLM, SMARCAL1, and PRIMPOL in ALT-positive cancer cells and its therapeutic implications. In SMARCAL1-proficient ALT-positive cancer cells (left), replication stress at telomeric DNA—initiated and coordinated by BLM activity—generates ssDNA intermediates that are efficiently processed through the concerted action of SMARCAL1, FANCM, and the BLM helicase complex. This tightly regulated pathway sustains productive ALT activity, preserves telomere integrity, and supports cancer cell proliferation. In contrast, in SMARCAL1-depleted ALT-positive cancer cells (right), BLM-dependent telomeric stress persists but cannot be properly resolved, leading to uncontrolled ssDNA accumulation exacerbated by aberrant PRIMPOL-mediated repriming. This deregulated processing renders ALT non-productive, amplifies telomeric stress, and triggers robust DNA damage response activation. The resulting cellular outcomes include senescence or reduced cancer cell growth, thereby compromising tumor homeostasis. Senescence induced by SMARCAL1 depletion can be exploited through the use of senolytic agents to enhance tumor clearance.

Article Snippet: In4mer arrays were ordered as individual eBlocks (IDT) and cloned into the lentiviral Cas12a gRNA expression backbone pRDA_052 (Addgene #136474) using the restriction cut site Esp3I.

Techniques: Fluorescence, Transfection, Quantitative RT-PCR, Expressing, Control, Transduction, Functional Assay, Activity Assay, Activation Assay