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sffv grna expression vectors  (Addgene inc)


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    Structured Review

    Addgene inc sffv grna expression vectors
    Sffv Grna Expression Vectors, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/grna+expression/XE14+Xbhh-N+(Plasmid+%2316994)/pmc12933725-971-0-5
    Average 93 stars, based on 8 article reviews
    sffv grna expression vectors - by Bioz Stars, 2026-10
    93/100 stars

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    Related Articles

    CRISPR:

    Article Title: Development of Degraders and 2-pyridinecarboxyaldehyde (2-PCA) as a recruitment Ligand for FBXO22
    Article Snippet: Dose-response curves were generated using Prism 10 (GraphPad), and IC50 values were calculated accordingly. .. The Bison CRISPR library targets 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes and contains 2,852 guide RNAs (Addgene #169942) . ..

    Article Title: Template-assisted covalent modification underlies activity of covalent molecular glues.
    Article Snippet: .. UPS-targeted BRD4BD2 reporter CRISPR screen The UPS-targeted CRISPR library (BISON sgRNA library; Addgene, 169942 (ref. 13)) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2,852 sgRNAs was cloned into the pXPR003 vector. ..

    Article Title: Template-assisted covalent modification underlies activity of covalent molecular glues
    Article Snippet: .. The UPS-targeted CRISPR library (BISON sgRNA library; Addgene, 169942 (ref. )) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2,852 sgRNAs was cloned into the pXPR003 vector. ..

    Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display
    Article Snippet: .. The Bison CRISPR library targets 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes and contains 2,852 guide RNAs (Addgene #169942) . ..

    Article Title: Discovery of electrophilic degraders that exploit SNAr chemistry
    Article Snippet: .. The Bison CRISPR library targets 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes and contains 2,852 guide RNAs (Addgene #169942) . ..

    Article Title: Chemical Specification of E3 Ubiquitin Ligase Engagement by Cysteine-Reactive Chemistry
    Article Snippet: .. UPS-targeted BRD4BD1 and BRD4BD2 reporter CRISPR screen The Ubiquitin Proteasome System (UPS)-targeted CRISPR library (BISON sgRNA library, Addgene 169942) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2852 sgRNAs was cloned into the pXPR003 vector. .. UPS-targeted BRD4BD1 and BRD4BD2 reporter CRISPR screen The Ubiquitin Proteasome System (UPS)-targeted CRISPR library (BISON sgRNA library, Addgene 169942) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2852 sgRNAs was cloned into the pXPR003 vector.

    Ubiquitin Proteomics:

    Article Title: Development of Degraders and 2-pyridinecarboxyaldehyde (2-PCA) as a recruitment Ligand for FBXO22
    Article Snippet: Dose-response curves were generated using Prism 10 (GraphPad), and IC50 values were calculated accordingly. .. The Bison CRISPR library targets 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes and contains 2,852 guide RNAs (Addgene #169942) . ..

    Article Title: Template-assisted covalent modification underlies activity of covalent molecular glues.
    Article Snippet: .. UPS-targeted BRD4BD2 reporter CRISPR screen The UPS-targeted CRISPR library (BISON sgRNA library; Addgene, 169942 (ref. 13)) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2,852 sgRNAs was cloned into the pXPR003 vector. ..

    Article Title: Template-assisted covalent modification underlies activity of covalent molecular glues
    Article Snippet: .. The UPS-targeted CRISPR library (BISON sgRNA library; Addgene, 169942 (ref. )) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2,852 sgRNAs was cloned into the pXPR003 vector. ..

    Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display
    Article Snippet: .. The Bison CRISPR library targets 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes and contains 2,852 guide RNAs (Addgene #169942) . ..

    Article Title: Discovery of electrophilic degraders that exploit SNAr chemistry
    Article Snippet: .. The Bison CRISPR library targets 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes and contains 2,852 guide RNAs (Addgene #169942) . ..

    Article Title: Chemical Specification of E3 Ubiquitin Ligase Engagement by Cysteine-Reactive Chemistry
    Article Snippet: .. UPS-targeted BRD4BD1 and BRD4BD2 reporter CRISPR screen The Ubiquitin Proteasome System (UPS)-targeted CRISPR library (BISON sgRNA library, Addgene 169942) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2852 sgRNAs was cloned into the pXPR003 vector. .. UPS-targeted BRD4BD1 and BRD4BD2 reporter CRISPR screen The Ubiquitin Proteasome System (UPS)-targeted CRISPR library (BISON sgRNA library, Addgene 169942) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2852 sgRNAs was cloned into the pXPR003 vector.

    Control:

    Article Title: Development of Degraders and 2-pyridinecarboxyaldehyde (2-PCA) as a recruitment Ligand for FBXO22
    Article Snippet: Dose-response curves were generated using Prism 10 (GraphPad), and IC50 values were calculated accordingly. .. The Bison CRISPR library targets 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes and contains 2,852 guide RNAs (Addgene #169942) . ..

    Article Title: Template-assisted covalent modification underlies activity of covalent molecular glues.
    Article Snippet: .. UPS-targeted BRD4BD2 reporter CRISPR screen The UPS-targeted CRISPR library (BISON sgRNA library; Addgene, 169942 (ref. 13)) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2,852 sgRNAs was cloned into the pXPR003 vector. ..

    Article Title: Template-assisted covalent modification underlies activity of covalent molecular glues
    Article Snippet: .. The UPS-targeted CRISPR library (BISON sgRNA library; Addgene, 169942 (ref. )) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2,852 sgRNAs was cloned into the pXPR003 vector. ..

    Article Title: Charged Molecular Glue Discovery Enabled by Targeted Degron Display
    Article Snippet: .. The Bison CRISPR library targets 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes and contains 2,852 guide RNAs (Addgene #169942) . ..

    Article Title: Discovery of electrophilic degraders that exploit SNAr chemistry
    Article Snippet: .. The Bison CRISPR library targets 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes and contains 2,852 guide RNAs (Addgene #169942) . ..

    Article Title: Chemical Specification of E3 Ubiquitin Ligase Engagement by Cysteine-Reactive Chemistry
    Article Snippet: .. UPS-targeted BRD4BD1 and BRD4BD2 reporter CRISPR screen The Ubiquitin Proteasome System (UPS)-targeted CRISPR library (BISON sgRNA library, Addgene 169942) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2852 sgRNAs was cloned into the pXPR003 vector. .. UPS-targeted BRD4BD1 and BRD4BD2 reporter CRISPR screen The Ubiquitin Proteasome System (UPS)-targeted CRISPR library (BISON sgRNA library, Addgene 169942) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2852 sgRNAs was cloned into the pXPR003 vector.

    Expressing:

    Article Title: Metabolic control of innate immune activation in TET2 -mutant clonal hematopoiesis
    Article Snippet: pInducer21 , Addgene; Meerbrey et al. 57 , Plasmid #46948. .. SFFV gRNA expression vectors , Addgene , Plasmids #169941 and #169940. .. pAAV-MCS , Cell Biolabs , Cat#VPK-411.

    Clone Assay:

    Article Title: Template-assisted covalent modification underlies activity of covalent molecular glues.
    Article Snippet: .. UPS-targeted BRD4BD2 reporter CRISPR screen The UPS-targeted CRISPR library (BISON sgRNA library; Addgene, 169942 (ref. 13)) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2,852 sgRNAs was cloned into the pXPR003 vector. ..

    Article Title: Template-assisted covalent modification underlies activity of covalent molecular glues
    Article Snippet: .. The UPS-targeted CRISPR library (BISON sgRNA library; Addgene, 169942 (ref. )) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2,852 sgRNAs was cloned into the pXPR003 vector. ..

    Article Title: Chemical Specification of E3 Ubiquitin Ligase Engagement by Cysteine-Reactive Chemistry
    Article Snippet: .. UPS-targeted BRD4BD1 and BRD4BD2 reporter CRISPR screen The Ubiquitin Proteasome System (UPS)-targeted CRISPR library (BISON sgRNA library, Addgene 169942) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2852 sgRNAs was cloned into the pXPR003 vector. .. UPS-targeted BRD4BD1 and BRD4BD2 reporter CRISPR screen The Ubiquitin Proteasome System (UPS)-targeted CRISPR library (BISON sgRNA library, Addgene 169942) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2852 sgRNAs was cloned into the pXPR003 vector.

    Plasmid Preparation:

    Article Title: Template-assisted covalent modification underlies activity of covalent molecular glues.
    Article Snippet: .. UPS-targeted BRD4BD2 reporter CRISPR screen The UPS-targeted CRISPR library (BISON sgRNA library; Addgene, 169942 (ref. 13)) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2,852 sgRNAs was cloned into the pXPR003 vector. ..

    Article Title: Template-assisted covalent modification underlies activity of covalent molecular glues
    Article Snippet: .. The UPS-targeted CRISPR library (BISON sgRNA library; Addgene, 169942 (ref. )) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2,852 sgRNAs was cloned into the pXPR003 vector. ..

    Article Title: Chemical Specification of E3 Ubiquitin Ligase Engagement by Cysteine-Reactive Chemistry
    Article Snippet: .. UPS-targeted BRD4BD1 and BRD4BD2 reporter CRISPR screen The Ubiquitin Proteasome System (UPS)-targeted CRISPR library (BISON sgRNA library, Addgene 169942) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2852 sgRNAs was cloned into the pXPR003 vector. .. UPS-targeted BRD4BD1 and BRD4BD2 reporter CRISPR screen The Ubiquitin Proteasome System (UPS)-targeted CRISPR library (BISON sgRNA library, Addgene 169942) targeting 713 E1, E2 and E3 ubiquitin ligases, deubiquitinases and control genes with a total of 2852 sgRNAs was cloned into the pXPR003 vector.



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    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.

    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

    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.

    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

    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.

    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

    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.

    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