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spcas9 sgrna  (Addgene inc)


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    Structured Review

    Addgene inc spcas9 sgrna
    Spcas9 Sgrna, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pbluescript/SpCas9_sgRNA_expression_in_pBluescript+(Plasmid+%23122089)/pmc13025988-4-0-2
    Average 93 stars, based on 12 article reviews
    spcas9 sgrna - by Bioz Stars, 2026-10
    93/100 stars

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    Related Articles

    other:

    Article Title: The Disruption of the HIV-1 Gag Start Codon via Editing Using MmCas12m-Dual Base Editor-Loaded Virus-like Particles
    Article Snippet: The nucleotide sequence of both fusion proteins ( ) was cloned into psPAX2 via SphI and XcmI sites instead of the original sequence. psgRNA-MmCas12m and psgRNA-dSpCas9 encode specific single-guide RNA under the control of the U6 promoter. psgRNA-MmCas12m and psgRNA-dSpCas9 were obtained based on a donor plasmid (#122089, Addgene) by replacing the original sequence with the target sequence ( ) via PciI and KpnI sites. pKW was created by converting the Kozak sequence from GCCACCAUG to GATATCAUG in phMGFP using specific primers for site-directed mutagenesis.

    Article Title: The Disruption of the HIV-1 Gag Start Codon via Editing Using MmCas12m-Dual Base Editor-Loaded Virus-like Particles
    Article Snippet: The nucleotide sequence of both fusion proteins (Supplementary Figures S3 and S4) was cloned into psPAX2 via SphI and XcmI sites instead of the original sequence. psgRNA-MmCas12m and psgRNA-dSpCas9 encode specific single-guide RNA under the control of the U6 promoter. psgRNA-MmCas12m and psgRNA-dSpCas9 were obtained based on a donor plasmid (#122089, Addgene) by replacing the original sequence with the target sequence (Supplementary Figure S5) via PciI and KpnI sites. pKW was created by converting the Kozak sequence from GCCACCAUG to GATATCAUG in phMGFP using specific primers for site-directed mutagenesis.

    Expressing:

    Article Title: Detecting Methylation Changes Induced by Prime Editing
    Article Snippet: .. To generate pegRNA expression plasmids, gblocks or PCR products including spacer sequences, scaffold sequences, and 3′ extension sequences (pegRNA products from IDT) were amplified with indicated primers using Phusion master mix (Thermo Fisher Scientific, Waltham, MA, USA), which were subsequently cloned into a custom vector (Addgene plasmid no. 122089, BfuAI- and EcoRI-digested) by the Gibson assembly method (NEB). ..

    Article Title: Detecting Methylation Changes Induced by Prime Editing.
    Article Snippet: .. To generate pegRNA expression plasmids, gblocks or PCR products including spacer sequences, scaffold sequences, and 3′ extension sequences (pegRNA products from IDT) were amplified with indicated primers using Phusion master mix (Thermo Fisher Scientific, Waltham, MA, USA), which were subsequently cloned into a custom vector (Addgene plasmid no. 122089, BfuAI- and EcoRI-digested) by the Gibson assembly method (NEB). ..

    Article Title: mRNA-engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in vivo
    Article Snippet: .. Briefly, gblocks (ordered from GenScript) containing a spacer-scaffold sequence (for sgRNAs) or a 3′ direct repeat (DR)-spacer sequence (for crRNAs) were cloned into U6 expression vectors (Addgene, #122089 and #217005) that had been linearized by BstXI and BlpI digestion, using the Gibson assembly method (New England Biolabs [NEB], #E2611L). ..

    Article Title: The Disruption of the HIV-1 Gag Start Codon via Editing Using MmCas12m-Dual Base Editor-Loaded Virus-like Particles
    Article Snippet: dSpCas9 (#201953, Addgene) , Mammalian expression plasmid of catalytically inactive SpCas9. .. SpCas9_sgRNA (#122089, Addgene) , U6 driven sgRNA expression vector for cloning own guides. .. phMGFP , Codes green fluorescent protein under the control of the CMV promoter.

    Polymerase Chain Reaction:

    Article Title: Detecting Methylation Changes Induced by Prime Editing
    Article Snippet: .. To generate pegRNA expression plasmids, gblocks or PCR products including spacer sequences, scaffold sequences, and 3′ extension sequences (pegRNA products from IDT) were amplified with indicated primers using Phusion master mix (Thermo Fisher Scientific, Waltham, MA, USA), which were subsequently cloned into a custom vector (Addgene plasmid no. 122089, BfuAI- and EcoRI-digested) by the Gibson assembly method (NEB). ..

    Article Title: Detecting Methylation Changes Induced by Prime Editing.
    Article Snippet: .. To generate pegRNA expression plasmids, gblocks or PCR products including spacer sequences, scaffold sequences, and 3′ extension sequences (pegRNA products from IDT) were amplified with indicated primers using Phusion master mix (Thermo Fisher Scientific, Waltham, MA, USA), which were subsequently cloned into a custom vector (Addgene plasmid no. 122089, BfuAI- and EcoRI-digested) by the Gibson assembly method (NEB). ..

    Amplification:

    Article Title: Detecting Methylation Changes Induced by Prime Editing
    Article Snippet: .. To generate pegRNA expression plasmids, gblocks or PCR products including spacer sequences, scaffold sequences, and 3′ extension sequences (pegRNA products from IDT) were amplified with indicated primers using Phusion master mix (Thermo Fisher Scientific, Waltham, MA, USA), which were subsequently cloned into a custom vector (Addgene plasmid no. 122089, BfuAI- and EcoRI-digested) by the Gibson assembly method (NEB). ..

    Article Title: Detecting Methylation Changes Induced by Prime Editing.
    Article Snippet: .. To generate pegRNA expression plasmids, gblocks or PCR products including spacer sequences, scaffold sequences, and 3′ extension sequences (pegRNA products from IDT) were amplified with indicated primers using Phusion master mix (Thermo Fisher Scientific, Waltham, MA, USA), which were subsequently cloned into a custom vector (Addgene plasmid no. 122089, BfuAI- and EcoRI-digested) by the Gibson assembly method (NEB). ..

    Clone Assay:

    Article Title: Detecting Methylation Changes Induced by Prime Editing
    Article Snippet: .. To generate pegRNA expression plasmids, gblocks or PCR products including spacer sequences, scaffold sequences, and 3′ extension sequences (pegRNA products from IDT) were amplified with indicated primers using Phusion master mix (Thermo Fisher Scientific, Waltham, MA, USA), which were subsequently cloned into a custom vector (Addgene plasmid no. 122089, BfuAI- and EcoRI-digested) by the Gibson assembly method (NEB). ..

    Article Title: Detecting Methylation Changes Induced by Prime Editing.
    Article Snippet: .. To generate pegRNA expression plasmids, gblocks or PCR products including spacer sequences, scaffold sequences, and 3′ extension sequences (pegRNA products from IDT) were amplified with indicated primers using Phusion master mix (Thermo Fisher Scientific, Waltham, MA, USA), which were subsequently cloned into a custom vector (Addgene plasmid no. 122089, BfuAI- and EcoRI-digested) by the Gibson assembly method (NEB). ..

    Article Title: mRNA-engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in vivo
    Article Snippet: .. Briefly, gblocks (ordered from GenScript) containing a spacer-scaffold sequence (for sgRNAs) or a 3′ direct repeat (DR)-spacer sequence (for crRNAs) were cloned into U6 expression vectors (Addgene, #122089 and #217005) that had been linearized by BstXI and BlpI digestion, using the Gibson assembly method (New England Biolabs [NEB], #E2611L). ..

    Article Title: Enhancing prime editing in hematopoietic stem and progenitor cells by modulating nucleotide metabolism.
    Article Snippet: Therapeutic prime editing of hematopoietic stem and progenitor cells (HSPCs) holds great potential to remedy blood disorders.. Quiescent cells have low nucleotide levels and resist retroviral infection, and it is possible that nucleotide metabolism could limit reverse transcription-mediated prime editing in HSPCs.. We demonstrate that deoxynucleoside supplementation and Vpx-mediated degradation of SAMHD1 improve prime editing efficiency in HSPCs, especially when coupled with editing approaches that evade mismatch repair.

    Plasmid Preparation:

    Article Title: Detecting Methylation Changes Induced by Prime Editing
    Article Snippet: .. To generate pegRNA expression plasmids, gblocks or PCR products including spacer sequences, scaffold sequences, and 3′ extension sequences (pegRNA products from IDT) were amplified with indicated primers using Phusion master mix (Thermo Fisher Scientific, Waltham, MA, USA), which were subsequently cloned into a custom vector (Addgene plasmid no. 122089, BfuAI- and EcoRI-digested) by the Gibson assembly method (NEB). ..

    Article Title: Detecting Methylation Changes Induced by Prime Editing.
    Article Snippet: .. To generate pegRNA expression plasmids, gblocks or PCR products including spacer sequences, scaffold sequences, and 3′ extension sequences (pegRNA products from IDT) were amplified with indicated primers using Phusion master mix (Thermo Fisher Scientific, Waltham, MA, USA), which were subsequently cloned into a custom vector (Addgene plasmid no. 122089, BfuAI- and EcoRI-digested) by the Gibson assembly method (NEB). ..

    Sequencing:

    Article Title: mRNA-engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in vivo
    Article Snippet: .. Briefly, gblocks (ordered from GenScript) containing a spacer-scaffold sequence (for sgRNAs) or a 3′ direct repeat (DR)-spacer sequence (for crRNAs) were cloned into U6 expression vectors (Addgene, #122089 and #217005) that had been linearized by BstXI and BlpI digestion, using the Gibson assembly method (New England Biolabs [NEB], #E2611L). ..

    Article Title: mRNA-Engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in Vivo
    Article Snippet: .. 86 MATERIALS AND METHODS 87 General methods and molecular cloning 88 Expression plasmids used for sgRNAs or crRNAs were constructed based on previously 89 described backbones.35 Briefly, gblocks (ordered from GenScript) containing a spacer-scaffold 90 sequence (for sgRNAs) or a 3’ Direct Repeat (DR)-spacer sequence (for crRNAs) were cloned 91 into U6 expression vectors (Addgene ##122089 and ##217005) that had been linearized by BstXI 92 and BlpI digestion, using the Gibson Assembly method (NEB, #E2611L). ..

    Molecular Cloning:

    Article Title: mRNA-Engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in Vivo
    Article Snippet: .. 86 MATERIALS AND METHODS 87 General methods and molecular cloning 88 Expression plasmids used for sgRNAs or crRNAs were constructed based on previously 89 described backbones.35 Briefly, gblocks (ordered from GenScript) containing a spacer-scaffold 90 sequence (for sgRNAs) or a 3’ Direct Repeat (DR)-spacer sequence (for crRNAs) were cloned 91 into U6 expression vectors (Addgene ##122089 and ##217005) that had been linearized by BstXI 92 and BlpI digestion, using the Gibson Assembly method (NEB, #E2611L). ..

    Construct:

    Article Title: mRNA-Engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in Vivo
    Article Snippet: .. 86 MATERIALS AND METHODS 87 General methods and molecular cloning 88 Expression plasmids used for sgRNAs or crRNAs were constructed based on previously 89 described backbones.35 Briefly, gblocks (ordered from GenScript) containing a spacer-scaffold 90 sequence (for sgRNAs) or a 3’ Direct Repeat (DR)-spacer sequence (for crRNAs) were cloned 91 into U6 expression vectors (Addgene ##122089 and ##217005) that had been linearized by BstXI 92 and BlpI digestion, using the Gibson Assembly method (NEB, #E2611L). ..

    Cloning:

    Article Title: The Disruption of the HIV-1 Gag Start Codon via Editing Using MmCas12m-Dual Base Editor-Loaded Virus-like Particles
    Article Snippet: dSpCas9 (#201953, Addgene) , Mammalian expression plasmid of catalytically inactive SpCas9. .. SpCas9_sgRNA (#122089, Addgene) , U6 driven sgRNA expression vector for cloning own guides. .. phMGFP , Codes green fluorescent protein under the control of the CMV promoter.



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    Image Search Results


    LJ3402 prevents hepatic senescence and mitochondrial dysfunction in aged mice. (A) Representative images of hematoxylin and eosin (H&E) (top), SA‐β‐gal (middle), and Oil Red O (bottom) staining on frozen liver sections from the indicated group. (B) The mRNA levels of marker genes involved in senescence ( p16 , p21 , p53 ) and protein levels (γH2AX, p21, p53) in the liver of young and aged mice were determined by RT‐qPCR or immunoblotting ( n = 3; three randomly selected samples per group). (C) Expression of marker genes involved in lipid metabolism ( Srebp1c , Pparα , Cpt1 ) and mitochondrial homeostasis ( Sirt1 , Pgc‐1α , NRF1 , TFAM ) in the liver was determined using RT‐qPCR. (D) Hepatic triglyceride (TG), total cholesterol (TC), and free fatty acid (FFA), and (E) plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ( n = 5; three randomly selected samples per group). (F) Livers separated from mice were incubated in culture medium, and the media were collected after 12 h and 24 h of incubation; the concentrations of TNF‐α and IL‐1β in the media were measured ( n = 5; three randomly selected samples per group). All data are presented as the mean ± SEM. * p < 0.05 and ** p < 0.01. Different lowercase letters above the bars indicate statistically significant differences ( p < 0.05).

    Journal: Biofactors (Oxford, England)

    Article Title: Lactobacillus johnsonii JNU3402 Ameliorates Age‐Related Liver Dysfunction Through Stimulating PGC ‐1α‐Mediated SIRT1 Expression

    doi: 10.1002/biof.70069

    Figure Lengend Snippet: LJ3402 prevents hepatic senescence and mitochondrial dysfunction in aged mice. (A) Representative images of hematoxylin and eosin (H&E) (top), SA‐β‐gal (middle), and Oil Red O (bottom) staining on frozen liver sections from the indicated group. (B) The mRNA levels of marker genes involved in senescence ( p16 , p21 , p53 ) and protein levels (γH2AX, p21, p53) in the liver of young and aged mice were determined by RT‐qPCR or immunoblotting ( n = 3; three randomly selected samples per group). (C) Expression of marker genes involved in lipid metabolism ( Srebp1c , Pparα , Cpt1 ) and mitochondrial homeostasis ( Sirt1 , Pgc‐1α , NRF1 , TFAM ) in the liver was determined using RT‐qPCR. (D) Hepatic triglyceride (TG), total cholesterol (TC), and free fatty acid (FFA), and (E) plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ( n = 5; three randomly selected samples per group). (F) Livers separated from mice were incubated in culture medium, and the media were collected after 12 h and 24 h of incubation; the concentrations of TNF‐α and IL‐1β in the media were measured ( n = 5; three randomly selected samples per group). All data are presented as the mean ± SEM. * p < 0.05 and ** p < 0.01. Different lowercase letters above the bars indicate statistically significant differences ( p < 0.05).

    Article Snippet: The pBluescript II KS(+)‐p21 promoter‐luc plasmid was purchased from Addgene (MA, USA) and designated as p21 promoter‐luc.

    Techniques: Staining, Marker, Quantitative RT-PCR, Western Blot, Expressing, Clinical Proteomics, Incubation

    SIRT1 modulates LJ3402‐mediated suppression of p53 function in senescent AML12 hepatocytes. (A) Acetylation of p53 after 5 days of treatment with LJ3402‐CM (LJ‐CM) and 50 μM sirtinol during H 2 O 2 ‐induced senescence. (A, C–I) Senescence of AML12 hepatocytes was induced by 1 mM or 750 μM H 2 O 2 for 1 h per day for 7 days. LJ‐CM, 50 μM sirtinol, or 20 μM PFTα were added on days 3–7 during H 2 O 2 exposure. (B) Luciferase reporter activity in HEK293T cells transfected with a reporter plasmid ( p21 promoter–luc) and a p53 expression plasmid. Twelve hours after transfection, cells were treated with LJ‐CM and 50 μM sirtinol for 24 h with or without 100 μM H 2 O 2 . (C) The mRNA and protein levels of p21, p53, PGC‐1α, and SIRT1 measured by RT‐qPCR and immunoblotting, respectively. (D) SA‐β‐gal staining. (E) mRNA levels of genes involved in mitochondrial biogenesis ( NRF1 and TFAM ) and oxidant defense ( CAT and SOD ). (F) ROS levels. (G) Oxygen consumption rates (OCRs) measured at baseline and after treatment with 2.5 μM oligomycin, 1 μM FCCP, or 1 μM rotenone/antimycin A. (H) Oil Red O staining. (I) Intracellular TG levels in senescent AML12 hepatocytes treated with LJ‐CM, sirtinol, and PFTα. All data are presented as the mean ± SEM. Different lowercase letters above the bars indicate statistically significant differences ( p < 0.05).

    Journal: Biofactors (Oxford, England)

    Article Title: Lactobacillus johnsonii JNU3402 Ameliorates Age‐Related Liver Dysfunction Through Stimulating PGC ‐1α‐Mediated SIRT1 Expression

    doi: 10.1002/biof.70069

    Figure Lengend Snippet: SIRT1 modulates LJ3402‐mediated suppression of p53 function in senescent AML12 hepatocytes. (A) Acetylation of p53 after 5 days of treatment with LJ3402‐CM (LJ‐CM) and 50 μM sirtinol during H 2 O 2 ‐induced senescence. (A, C–I) Senescence of AML12 hepatocytes was induced by 1 mM or 750 μM H 2 O 2 for 1 h per day for 7 days. LJ‐CM, 50 μM sirtinol, or 20 μM PFTα were added on days 3–7 during H 2 O 2 exposure. (B) Luciferase reporter activity in HEK293T cells transfected with a reporter plasmid ( p21 promoter–luc) and a p53 expression plasmid. Twelve hours after transfection, cells were treated with LJ‐CM and 50 μM sirtinol for 24 h with or without 100 μM H 2 O 2 . (C) The mRNA and protein levels of p21, p53, PGC‐1α, and SIRT1 measured by RT‐qPCR and immunoblotting, respectively. (D) SA‐β‐gal staining. (E) mRNA levels of genes involved in mitochondrial biogenesis ( NRF1 and TFAM ) and oxidant defense ( CAT and SOD ). (F) ROS levels. (G) Oxygen consumption rates (OCRs) measured at baseline and after treatment with 2.5 μM oligomycin, 1 μM FCCP, or 1 μM rotenone/antimycin A. (H) Oil Red O staining. (I) Intracellular TG levels in senescent AML12 hepatocytes treated with LJ‐CM, sirtinol, and PFTα. All data are presented as the mean ± SEM. Different lowercase letters above the bars indicate statistically significant differences ( p < 0.05).

    Article Snippet: The pBluescript II KS(+)‐p21 promoter‐luc plasmid was purchased from Addgene (MA, USA) and designated as p21 promoter‐luc.

    Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Staining