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spcas9 coding sequence  (Addgene inc)


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    Addgene inc spcas9 coding sequence
    Spcas9 Coding Sequence, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 1650 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/spcas9+sequence/lentiCas9-Blast+(Plasmid+%2352962)/pmc12958575-257-6-13
    Average 96 stars, based on 1650 article reviews
    spcas9 coding sequence - by Bioz Stars, 2026-09
    96/100 stars

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    Related Articles

    Over Expression:

    Article Title: SOX17 enables immune evasion of early colorectal adenomas and cancers
    Article Snippet: To knockout Ifngr1, H2k1 , B2m, and Cxcl10 in SOX17KO organoids, sgRNAs for Ifngr1 (5ʹ-CGACTTCAGGGTGAAATACG-3ʹ), H2k1 (5ʹ-CGAGATATGAGCCGCGGGCG-3ʹ), B2m (5ʹ-AGTATACTCACGCCACCCAC-3ʹ) and Cxcl10 (5ʹ-AGCGGACCGTCCTTGCGAGA-3ʹ) were cloned into LentiCRISPRv2 neo (addgene, #98292) and the analogous methods (except that organoids were selected by 400 μg/ml G418 (Sigma-Aldrich, G8168)) were used to establish the knockout clones. .. For constitutive SOX17 overexpression, mouse Sox17 sequence was amplified by PCR from FUW-TetO-SOX17 plasmid (addgene, #128828) to have overlapping DNA ends with the donor EF-1a-spCas9-T2A-mNeonGreen-P2A-puro vector (addgene, #122183). spCas9 sequence of the donor EF-1a-spCas9-T2A-mNeonGreen-P2A-puro vector was cut by restriction enzymes and replaced with mouse SOX17 sequence using In-Fusion HD Cloning Kit (Clontech, 638916) following the manufacturer’s protocol. ..

    Sequencing:

    Article Title: SOX17 enables immune evasion of early colorectal adenomas and cancers
    Article Snippet: To knockout Ifngr1, H2k1 , B2m, and Cxcl10 in SOX17KO organoids, sgRNAs for Ifngr1 (5ʹ-CGACTTCAGGGTGAAATACG-3ʹ), H2k1 (5ʹ-CGAGATATGAGCCGCGGGCG-3ʹ), B2m (5ʹ-AGTATACTCACGCCACCCAC-3ʹ) and Cxcl10 (5ʹ-AGCGGACCGTCCTTGCGAGA-3ʹ) were cloned into LentiCRISPRv2 neo (addgene, #98292) and the analogous methods (except that organoids were selected by 400 μg/ml G418 (Sigma-Aldrich, G8168)) were used to establish the knockout clones. .. For constitutive SOX17 overexpression, mouse Sox17 sequence was amplified by PCR from FUW-TetO-SOX17 plasmid (addgene, #128828) to have overlapping DNA ends with the donor EF-1a-spCas9-T2A-mNeonGreen-P2A-puro vector (addgene, #122183). spCas9 sequence of the donor EF-1a-spCas9-T2A-mNeonGreen-P2A-puro vector was cut by restriction enzymes and replaced with mouse SOX17 sequence using In-Fusion HD Cloning Kit (Clontech, 638916) following the manufacturer’s protocol. ..

    Article Title: A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa
    Article Snippet: The strain NcCas9SG was created by transforming N. crassa strain FGSC #6103 (his-3 (Y234M723) mat A) with the vector construct pSG897 carrying the Spcas9 sequence with a 3′ nucleoplasmin NLS. .. The Spcas9 sequence was amplified from the plasmid pSPCas9(BB)-2A-GFP (Addgene plasmid ID: 48138) using the oligonucleotides SG3449 and SG3444 adding an AscI and SwaI recognition site to the PCR product. .. Blunt-end PCR products were subcloned using a CloneJet PCR cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendation.

    Article Title: DNA polymerase λ-driven targeted mutagenesis for directed evolution in human cells
    Article Snippet: To generate CRISPR-Pol- E.coli , pcDNA3-CRISPR-Pol-RT and pcDNA3-CRISPR-λ constructs pcDNA3 plasmid was modified through GeneStrand cloning (Eurofins, see Supplementary Table S1 ) to insert additional restriction cloning sites (NheI, BamHI, AflII). .. The SpCas9 sequence was amplified from Addgene Plasmid 112095 and cloned using EcoRI and NheI. .. Human DNA Polymerase λ Δ2 was kindly provided by professor Emanuele Crespan (Istituto di Genetica Molecolare “Luigi Luca Cavalli-Sforza”, Pavia) and HIV-1 RT from Δ8.91 lentiviral packaging plasmid and cloned using BamHI and AflII. pIRES-EGFP-puro STOP, IN-661, IN-664, IN-639 and IN-636 alongside pRosetta-STOP (expressing the blasticidin-S deaminase gene, BSD ) plasmids were generated through site directed mutagenesis ( Supplementary Table S2 ).

    Article Title: Diverse anti-defence systems are encoded in the leading region of plasmids.
    Article Snippet: .. The SpCas9 sequence was amplified from Addgene plasmid no. 101044, followed by cloning into the pD5 vector using Gibson Assembly. ..

    Article Title: A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa.
    Article Snippet: Generation of N. crassa Cas9 strain NcCas9SG The strain NcCas9SG was created by transforming N. crassa strain FGSC #6103 (his-3 (Y234M723) mat A) with the vector construct pSG897 carrying the Spcas9 sequence with a 3′ nucleoplasmin NLS. .. The Spcas9 sequence was amplified from the plasmid pSPCas9(BB)-2A-GFP (Addgene plasmid ID: 48138) using the oligonucleotides SG3449 and SG3444 adding an AscI and SwaI recognition site to the PCR product. .. Blunt-end PCR products were subcloned using a CloneJet PCR cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendation.

    Article Title: Diverse anti-defence systems are encoded in the leading region of plasmids
    Article Snippet: .. The SpCas9 sequence was amplified from Addgene plasmid no. 101044, followed by cloning into the pD5 vector using Gibson Assembly. ..

    Article Title: Methods and compositions comprising CRISPR-Cpf1 and paired guide CRISPR RNAs for programmable genomic deletions
    Article Snippet: .. The lentiCRISPRv2 backbone used here contains a sgRNA expressing cassette (hU6 promoter and sgRNA) and SpCas9 sequence (Addgene plasmid 52961) (Sanjana, 2014). ..

    Amplification:

    Article Title: SOX17 enables immune evasion of early colorectal adenomas and cancers
    Article Snippet: To knockout Ifngr1, H2k1 , B2m, and Cxcl10 in SOX17KO organoids, sgRNAs for Ifngr1 (5ʹ-CGACTTCAGGGTGAAATACG-3ʹ), H2k1 (5ʹ-CGAGATATGAGCCGCGGGCG-3ʹ), B2m (5ʹ-AGTATACTCACGCCACCCAC-3ʹ) and Cxcl10 (5ʹ-AGCGGACCGTCCTTGCGAGA-3ʹ) were cloned into LentiCRISPRv2 neo (addgene, #98292) and the analogous methods (except that organoids were selected by 400 μg/ml G418 (Sigma-Aldrich, G8168)) were used to establish the knockout clones. .. For constitutive SOX17 overexpression, mouse Sox17 sequence was amplified by PCR from FUW-TetO-SOX17 plasmid (addgene, #128828) to have overlapping DNA ends with the donor EF-1a-spCas9-T2A-mNeonGreen-P2A-puro vector (addgene, #122183). spCas9 sequence of the donor EF-1a-spCas9-T2A-mNeonGreen-P2A-puro vector was cut by restriction enzymes and replaced with mouse SOX17 sequence using In-Fusion HD Cloning Kit (Clontech, 638916) following the manufacturer’s protocol. ..

    Article Title: A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa
    Article Snippet: The strain NcCas9SG was created by transforming N. crassa strain FGSC #6103 (his-3 (Y234M723) mat A) with the vector construct pSG897 carrying the Spcas9 sequence with a 3′ nucleoplasmin NLS. .. The Spcas9 sequence was amplified from the plasmid pSPCas9(BB)-2A-GFP (Addgene plasmid ID: 48138) using the oligonucleotides SG3449 and SG3444 adding an AscI and SwaI recognition site to the PCR product. .. Blunt-end PCR products were subcloned using a CloneJet PCR cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendation.

    Article Title: DNA polymerase λ-driven targeted mutagenesis for directed evolution in human cells
    Article Snippet: To generate CRISPR-Pol- E.coli , pcDNA3-CRISPR-Pol-RT and pcDNA3-CRISPR-λ constructs pcDNA3 plasmid was modified through GeneStrand cloning (Eurofins, see Supplementary Table S1 ) to insert additional restriction cloning sites (NheI, BamHI, AflII). .. The SpCas9 sequence was amplified from Addgene Plasmid 112095 and cloned using EcoRI and NheI. .. Human DNA Polymerase λ Δ2 was kindly provided by professor Emanuele Crespan (Istituto di Genetica Molecolare “Luigi Luca Cavalli-Sforza”, Pavia) and HIV-1 RT from Δ8.91 lentiviral packaging plasmid and cloned using BamHI and AflII. pIRES-EGFP-puro STOP, IN-661, IN-664, IN-639 and IN-636 alongside pRosetta-STOP (expressing the blasticidin-S deaminase gene, BSD ) plasmids were generated through site directed mutagenesis ( Supplementary Table S2 ).

    Article Title: Diverse anti-defence systems are encoded in the leading region of plasmids.
    Article Snippet: .. The SpCas9 sequence was amplified from Addgene plasmid no. 101044, followed by cloning into the pD5 vector using Gibson Assembly. ..

    Article Title: A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa.
    Article Snippet: Generation of N. crassa Cas9 strain NcCas9SG The strain NcCas9SG was created by transforming N. crassa strain FGSC #6103 (his-3 (Y234M723) mat A) with the vector construct pSG897 carrying the Spcas9 sequence with a 3′ nucleoplasmin NLS. .. The Spcas9 sequence was amplified from the plasmid pSPCas9(BB)-2A-GFP (Addgene plasmid ID: 48138) using the oligonucleotides SG3449 and SG3444 adding an AscI and SwaI recognition site to the PCR product. .. Blunt-end PCR products were subcloned using a CloneJet PCR cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendation.

    Article Title: Diverse anti-defence systems are encoded in the leading region of plasmids
    Article Snippet: .. The SpCas9 sequence was amplified from Addgene plasmid no. 101044, followed by cloning into the pD5 vector using Gibson Assembly. ..

    Polymerase Chain Reaction:

    Article Title: SOX17 enables immune evasion of early colorectal adenomas and cancers
    Article Snippet: To knockout Ifngr1, H2k1 , B2m, and Cxcl10 in SOX17KO organoids, sgRNAs for Ifngr1 (5ʹ-CGACTTCAGGGTGAAATACG-3ʹ), H2k1 (5ʹ-CGAGATATGAGCCGCGGGCG-3ʹ), B2m (5ʹ-AGTATACTCACGCCACCCAC-3ʹ) and Cxcl10 (5ʹ-AGCGGACCGTCCTTGCGAGA-3ʹ) were cloned into LentiCRISPRv2 neo (addgene, #98292) and the analogous methods (except that organoids were selected by 400 μg/ml G418 (Sigma-Aldrich, G8168)) were used to establish the knockout clones. .. For constitutive SOX17 overexpression, mouse Sox17 sequence was amplified by PCR from FUW-TetO-SOX17 plasmid (addgene, #128828) to have overlapping DNA ends with the donor EF-1a-spCas9-T2A-mNeonGreen-P2A-puro vector (addgene, #122183). spCas9 sequence of the donor EF-1a-spCas9-T2A-mNeonGreen-P2A-puro vector was cut by restriction enzymes and replaced with mouse SOX17 sequence using In-Fusion HD Cloning Kit (Clontech, 638916) following the manufacturer’s protocol. ..

    Article Title: A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa
    Article Snippet: The strain NcCas9SG was created by transforming N. crassa strain FGSC #6103 (his-3 (Y234M723) mat A) with the vector construct pSG897 carrying the Spcas9 sequence with a 3′ nucleoplasmin NLS. .. The Spcas9 sequence was amplified from the plasmid pSPCas9(BB)-2A-GFP (Addgene plasmid ID: 48138) using the oligonucleotides SG3449 and SG3444 adding an AscI and SwaI recognition site to the PCR product. .. Blunt-end PCR products were subcloned using a CloneJet PCR cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendation.

    Article Title: A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa.
    Article Snippet: Generation of N. crassa Cas9 strain NcCas9SG The strain NcCas9SG was created by transforming N. crassa strain FGSC #6103 (his-3 (Y234M723) mat A) with the vector construct pSG897 carrying the Spcas9 sequence with a 3′ nucleoplasmin NLS. .. The Spcas9 sequence was amplified from the plasmid pSPCas9(BB)-2A-GFP (Addgene plasmid ID: 48138) using the oligonucleotides SG3449 and SG3444 adding an AscI and SwaI recognition site to the PCR product. .. Blunt-end PCR products were subcloned using a CloneJet PCR cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendation.

    Plasmid Preparation:

    Article Title: SOX17 enables immune evasion of early colorectal adenomas and cancers
    Article Snippet: To knockout Ifngr1, H2k1 , B2m, and Cxcl10 in SOX17KO organoids, sgRNAs for Ifngr1 (5ʹ-CGACTTCAGGGTGAAATACG-3ʹ), H2k1 (5ʹ-CGAGATATGAGCCGCGGGCG-3ʹ), B2m (5ʹ-AGTATACTCACGCCACCCAC-3ʹ) and Cxcl10 (5ʹ-AGCGGACCGTCCTTGCGAGA-3ʹ) were cloned into LentiCRISPRv2 neo (addgene, #98292) and the analogous methods (except that organoids were selected by 400 μg/ml G418 (Sigma-Aldrich, G8168)) were used to establish the knockout clones. .. For constitutive SOX17 overexpression, mouse Sox17 sequence was amplified by PCR from FUW-TetO-SOX17 plasmid (addgene, #128828) to have overlapping DNA ends with the donor EF-1a-spCas9-T2A-mNeonGreen-P2A-puro vector (addgene, #122183). spCas9 sequence of the donor EF-1a-spCas9-T2A-mNeonGreen-P2A-puro vector was cut by restriction enzymes and replaced with mouse SOX17 sequence using In-Fusion HD Cloning Kit (Clontech, 638916) following the manufacturer’s protocol. ..

    Article Title: A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa
    Article Snippet: The strain NcCas9SG was created by transforming N. crassa strain FGSC #6103 (his-3 (Y234M723) mat A) with the vector construct pSG897 carrying the Spcas9 sequence with a 3′ nucleoplasmin NLS. .. The Spcas9 sequence was amplified from the plasmid pSPCas9(BB)-2A-GFP (Addgene plasmid ID: 48138) using the oligonucleotides SG3449 and SG3444 adding an AscI and SwaI recognition site to the PCR product. .. Blunt-end PCR products were subcloned using a CloneJet PCR cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendation.

    Article Title: DNA polymerase λ-driven targeted mutagenesis for directed evolution in human cells
    Article Snippet: To generate CRISPR-Pol- E.coli , pcDNA3-CRISPR-Pol-RT and pcDNA3-CRISPR-λ constructs pcDNA3 plasmid was modified through GeneStrand cloning (Eurofins, see Supplementary Table S1 ) to insert additional restriction cloning sites (NheI, BamHI, AflII). .. The SpCas9 sequence was amplified from Addgene Plasmid 112095 and cloned using EcoRI and NheI. .. Human DNA Polymerase λ Δ2 was kindly provided by professor Emanuele Crespan (Istituto di Genetica Molecolare “Luigi Luca Cavalli-Sforza”, Pavia) and HIV-1 RT from Δ8.91 lentiviral packaging plasmid and cloned using BamHI and AflII. pIRES-EGFP-puro STOP, IN-661, IN-664, IN-639 and IN-636 alongside pRosetta-STOP (expressing the blasticidin-S deaminase gene, BSD ) plasmids were generated through site directed mutagenesis ( Supplementary Table S2 ).

    Article Title: Diverse anti-defence systems are encoded in the leading region of plasmids.
    Article Snippet: .. The SpCas9 sequence was amplified from Addgene plasmid no. 101044, followed by cloning into the pD5 vector using Gibson Assembly. ..

    Article Title: A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa.
    Article Snippet: Generation of N. crassa Cas9 strain NcCas9SG The strain NcCas9SG was created by transforming N. crassa strain FGSC #6103 (his-3 (Y234M723) mat A) with the vector construct pSG897 carrying the Spcas9 sequence with a 3′ nucleoplasmin NLS. .. The Spcas9 sequence was amplified from the plasmid pSPCas9(BB)-2A-GFP (Addgene plasmid ID: 48138) using the oligonucleotides SG3449 and SG3444 adding an AscI and SwaI recognition site to the PCR product. .. Blunt-end PCR products were subcloned using a CloneJet PCR cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendation.

    Article Title: Diverse anti-defence systems are encoded in the leading region of plasmids
    Article Snippet: .. The SpCas9 sequence was amplified from Addgene plasmid no. 101044, followed by cloning into the pD5 vector using Gibson Assembly. ..

    Article Title: Methods and compositions comprising CRISPR-Cpf1 and paired guide CRISPR RNAs for programmable genomic deletions
    Article Snippet: .. The lentiCRISPRv2 backbone used here contains a sgRNA expressing cassette (hU6 promoter and sgRNA) and SpCas9 sequence (Addgene plasmid 52961) (Sanjana, 2014). ..

    Cloning:

    Article Title: SOX17 enables immune evasion of early colorectal adenomas and cancers
    Article Snippet: To knockout Ifngr1, H2k1 , B2m, and Cxcl10 in SOX17KO organoids, sgRNAs for Ifngr1 (5ʹ-CGACTTCAGGGTGAAATACG-3ʹ), H2k1 (5ʹ-CGAGATATGAGCCGCGGGCG-3ʹ), B2m (5ʹ-AGTATACTCACGCCACCCAC-3ʹ) and Cxcl10 (5ʹ-AGCGGACCGTCCTTGCGAGA-3ʹ) were cloned into LentiCRISPRv2 neo (addgene, #98292) and the analogous methods (except that organoids were selected by 400 μg/ml G418 (Sigma-Aldrich, G8168)) were used to establish the knockout clones. .. For constitutive SOX17 overexpression, mouse Sox17 sequence was amplified by PCR from FUW-TetO-SOX17 plasmid (addgene, #128828) to have overlapping DNA ends with the donor EF-1a-spCas9-T2A-mNeonGreen-P2A-puro vector (addgene, #122183). spCas9 sequence of the donor EF-1a-spCas9-T2A-mNeonGreen-P2A-puro vector was cut by restriction enzymes and replaced with mouse SOX17 sequence using In-Fusion HD Cloning Kit (Clontech, 638916) following the manufacturer’s protocol. ..

    Article Title: Diverse anti-defence systems are encoded in the leading region of plasmids.
    Article Snippet: .. The SpCas9 sequence was amplified from Addgene plasmid no. 101044, followed by cloning into the pD5 vector using Gibson Assembly. ..

    Article Title: Diverse anti-defence systems are encoded in the leading region of plasmids
    Article Snippet: .. The SpCas9 sequence was amplified from Addgene plasmid no. 101044, followed by cloning into the pD5 vector using Gibson Assembly. ..

    Clone Assay:

    Article Title: DNA polymerase λ-driven targeted mutagenesis for directed evolution in human cells
    Article Snippet: To generate CRISPR-Pol- E.coli , pcDNA3-CRISPR-Pol-RT and pcDNA3-CRISPR-λ constructs pcDNA3 plasmid was modified through GeneStrand cloning (Eurofins, see Supplementary Table S1 ) to insert additional restriction cloning sites (NheI, BamHI, AflII). .. The SpCas9 sequence was amplified from Addgene Plasmid 112095 and cloned using EcoRI and NheI. .. Human DNA Polymerase λ Δ2 was kindly provided by professor Emanuele Crespan (Istituto di Genetica Molecolare “Luigi Luca Cavalli-Sforza”, Pavia) and HIV-1 RT from Δ8.91 lentiviral packaging plasmid and cloned using BamHI and AflII. pIRES-EGFP-puro STOP, IN-661, IN-664, IN-639 and IN-636 alongside pRosetta-STOP (expressing the blasticidin-S deaminase gene, BSD ) plasmids were generated through site directed mutagenesis ( Supplementary Table S2 ).

    Expressing:

    Article Title: Methods and compositions comprising CRISPR-Cpf1 and paired guide CRISPR RNAs for programmable genomic deletions
    Article Snippet: .. The lentiCRISPRv2 backbone used here contains a sgRNA expressing cassette (hU6 promoter and sgRNA) and SpCas9 sequence (Addgene plasmid 52961) (Sanjana, 2014). ..



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    Image Search Results


    Fig. 2 The modified 3TC scaffold boosts SpCas9 gRNA expression levels compared to the original 4T scaffold. (A) DNA sequence of the 4T and modified 3TC scaffolds. (B) Relative quantification (RQ) of mDmd gRNA delivered by nucleofection of PX459.V2 (4T) to C2C12 cells, by qRT-PCR. Mean Log2RQ ± 95% CI; n = 3. One-way ANOVA with Dunnett’s multiple comparisons test was performed on ΔΔCT values, **p ≤ 0.01, ***p ≤ 0.001. (C, D, E) Comparison of the relative quantities of mDmd Sp gRNA, delivered by PX459.V2, pdg459.V2 (2 × 4T) and PX459.V3 (3TC), measured by qRT-PCR. Mean Log2RQ ± 95% CI; n = 3. One-way ANOVA with Tukey’s multiple comparisons test performed on ΔΔCT values, **p ≤ 0.01, ***p ≤ 0.001

    Journal: BMC genomics

    Article Title: Optimal SpCas9- and SaCas9-mediated gene editing by enhancing gRNA transcript levels through scaffold poly-T tract reduction.

    doi: 10.1186/s12864-025-11317-2

    Figure Lengend Snippet: Fig. 2 The modified 3TC scaffold boosts SpCas9 gRNA expression levels compared to the original 4T scaffold. (A) DNA sequence of the 4T and modified 3TC scaffolds. (B) Relative quantification (RQ) of mDmd gRNA delivered by nucleofection of PX459.V2 (4T) to C2C12 cells, by qRT-PCR. Mean Log2RQ ± 95% CI; n = 3. One-way ANOVA with Dunnett’s multiple comparisons test was performed on ΔΔCT values, **p ≤ 0.01, ***p ≤ 0.001. (C, D, E) Comparison of the relative quantities of mDmd Sp gRNA, delivered by PX459.V2, pdg459.V2 (2 × 4T) and PX459.V3 (3TC), measured by qRT-PCR. Mean Log2RQ ± 95% CI; n = 3. One-way ANOVA with Tukey’s multiple comparisons test performed on ΔΔCT values, **p ≤ 0.01, ***p ≤ 0.001

    Article Snippet: SaCas9Puro.V2 was generated by replacing the SpCas9 coding sequences of PX459.V2 with SaCas9 coding sequences from PX601 (Addgene #61591).

    Techniques: Modification, Expressing, Sequencing, Quantitative Proteomics, Quantitative RT-PCR, Comparison

    Fig. 5 Editing efficiencies of high-fidelity SpCas9s with the 3TC scaffold. Comparison of PX459.V2 SpCas9-HF1 (4T), PX459.V3 SpCas9-HF1 (3TC), PX459.V2 eSpCas9(1.1) (4T) and PX459.V3 eSpCas9(1.1) (3TC) plasmids de livered by lipofection at a (A) high and (B) low plasmid dose without puro mycin selection in HEK239T cells, assessed by deep amplicon sequencing. Mean ± SEM; n = 3. Two-way ANOVA with Šídák’s multiple comparisons test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (C) Editing efficiencies of hDMD-B in the G19 gRNA configuration with WT and high-fidelity Sp-Cas9 plasmids delivered by nucleofection with puromycin selection in HEK293Ts. Two- way ANOVA with Šídák’s multiple comparisons test; *p ≤ 0.05, ***p ≤ 0.001

    Journal: BMC genomics

    Article Title: Optimal SpCas9- and SaCas9-mediated gene editing by enhancing gRNA transcript levels through scaffold poly-T tract reduction.

    doi: 10.1186/s12864-025-11317-2

    Figure Lengend Snippet: Fig. 5 Editing efficiencies of high-fidelity SpCas9s with the 3TC scaffold. Comparison of PX459.V2 SpCas9-HF1 (4T), PX459.V3 SpCas9-HF1 (3TC), PX459.V2 eSpCas9(1.1) (4T) and PX459.V3 eSpCas9(1.1) (3TC) plasmids de livered by lipofection at a (A) high and (B) low plasmid dose without puro mycin selection in HEK239T cells, assessed by deep amplicon sequencing. Mean ± SEM; n = 3. Two-way ANOVA with Šídák’s multiple comparisons test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (C) Editing efficiencies of hDMD-B in the G19 gRNA configuration with WT and high-fidelity Sp-Cas9 plasmids delivered by nucleofection with puromycin selection in HEK293Ts. Two- way ANOVA with Šídák’s multiple comparisons test; *p ≤ 0.05, ***p ≤ 0.001

    Article Snippet: SaCas9Puro.V2 was generated by replacing the SpCas9 coding sequences of PX459.V2 with SaCas9 coding sequences from PX601 (Addgene #61591).

    Techniques: Comparison, Plasmid Preparation, Selection, Amplification, Sequencing