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spycas9 coding sequence  (Addgene inc)


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    Structured Review

    Addgene inc spycas9 coding sequence
    Marked increase in indel efficiency of the Cas9-RecJ and Cas9-GFP chimeric fusion proteins. ( A ) Schematic of a translational fusion of a modifier protein to <t>SpyCas9.</t> His tag (6X His) for protein purification and nuclear localization signal (NLS) were placed. ( B ) Comparison of the DNA cleavage activity of SpyCas9 and its chimeric proteins, C-terminal chimeric SpyCas9 translational fusion proteins with the nucleases RecJ, RecE, T5, lambda, mung bean exonuclease, and human terminal deoxynucleotidyl transferase (TdT), and with green fluorescent protein (GFP). The modifier proteins are described in Supplementary Table . The CCR5 DNA was digested with the purified enzymes and resolved on an agarose gel. Successful digestion cuts the 1.5 kb substrate DNA into two 0.75 kb DNAs. ( C ) DNA band intensities in the gel image. Statistical comparison was made between C9 and each of the C9R or C9G proteins. Significance was indicated on top of each bar. One-way ANOVA using Tukey’s multiple-comparison test. The fold change relative to C9 is indicated above each bar. ( D ) DNA insertion and deletion (Indel) efficiency of C9, C9R, and C9G for the CCR5 gene in HEK293T cells at 8, 16, and 24 h after RNP transfection as measured by a T7E1 assay. Averages from the three replicate experiments are plotted. Two-way ANOVA using Tukey’s multiple-comparison test. ( E ) Indel efficiency of C9, C9R, and C9G for the CCR5 , HPRT1 , and EMX1 genes (left to right) in HEK293T cells. At 24 h ( CCR5 and HPRT1 ) and 8 h ( EMX1 ) post-transfection, cells were harvested and subjected to targeted deep sequencing. The experiments were performed in triplicate. NT group represent the NGS data from non-treated control. One-way ANOVA using Tukey’s multiple-comparison test was performed using C9 data as control. ( F ) Distribution of the DNA deletion sizes after treatment with C9, C9R, and C9G. The number of deletion events around the protospacer site in the CCR5 gene was counted according to the 15 bp deletion intervals from targeted deep sequencing data (n = 3) data. ( G ) Distribution of DNA insertion sizes after treatment with C9, C9R, and C9G in the CCR5 gene. The number of insertion events was counted according to the 1 bp insertion intervals from targeted deep sequencing data (n = 3) data. 6–10 and 11–61 bp insertion intervals were also shown to include events with smaller numbers. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, and n.s. indicates not significant.
    Spycas9 Coding Sequence, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/spcas9+ng+coding+sequence/SpCas9-NG+(Plasmid+%23138566)/pmc08355345-119-1-4
    Average 93 stars, based on 27 article reviews
    spycas9 coding sequence - by Bioz Stars, 2026-10
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    1) Product Images from "Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins"

    Article Title: Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-95406-8

    Marked increase in indel efficiency of the Cas9-RecJ and Cas9-GFP chimeric fusion proteins. ( A ) Schematic of a translational fusion of a modifier protein to SpyCas9. His tag (6X His) for protein purification and nuclear localization signal (NLS) were placed. ( B ) Comparison of the DNA cleavage activity of SpyCas9 and its chimeric proteins, C-terminal chimeric SpyCas9 translational fusion proteins with the nucleases RecJ, RecE, T5, lambda, mung bean exonuclease, and human terminal deoxynucleotidyl transferase (TdT), and with green fluorescent protein (GFP). The modifier proteins are described in Supplementary Table . The CCR5 DNA was digested with the purified enzymes and resolved on an agarose gel. Successful digestion cuts the 1.5 kb substrate DNA into two 0.75 kb DNAs. ( C ) DNA band intensities in the gel image. Statistical comparison was made between C9 and each of the C9R or C9G proteins. Significance was indicated on top of each bar. One-way ANOVA using Tukey’s multiple-comparison test. The fold change relative to C9 is indicated above each bar. ( D ) DNA insertion and deletion (Indel) efficiency of C9, C9R, and C9G for the CCR5 gene in HEK293T cells at 8, 16, and 24 h after RNP transfection as measured by a T7E1 assay. Averages from the three replicate experiments are plotted. Two-way ANOVA using Tukey’s multiple-comparison test. ( E ) Indel efficiency of C9, C9R, and C9G for the CCR5 , HPRT1 , and EMX1 genes (left to right) in HEK293T cells. At 24 h ( CCR5 and HPRT1 ) and 8 h ( EMX1 ) post-transfection, cells were harvested and subjected to targeted deep sequencing. The experiments were performed in triplicate. NT group represent the NGS data from non-treated control. One-way ANOVA using Tukey’s multiple-comparison test was performed using C9 data as control. ( F ) Distribution of the DNA deletion sizes after treatment with C9, C9R, and C9G. The number of deletion events around the protospacer site in the CCR5 gene was counted according to the 15 bp deletion intervals from targeted deep sequencing data (n = 3) data. ( G ) Distribution of DNA insertion sizes after treatment with C9, C9R, and C9G in the CCR5 gene. The number of insertion events was counted according to the 1 bp insertion intervals from targeted deep sequencing data (n = 3) data. 6–10 and 11–61 bp insertion intervals were also shown to include events with smaller numbers. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, and n.s. indicates not significant.
    Figure Legend Snippet: Marked increase in indel efficiency of the Cas9-RecJ and Cas9-GFP chimeric fusion proteins. ( A ) Schematic of a translational fusion of a modifier protein to SpyCas9. His tag (6X His) for protein purification and nuclear localization signal (NLS) were placed. ( B ) Comparison of the DNA cleavage activity of SpyCas9 and its chimeric proteins, C-terminal chimeric SpyCas9 translational fusion proteins with the nucleases RecJ, RecE, T5, lambda, mung bean exonuclease, and human terminal deoxynucleotidyl transferase (TdT), and with green fluorescent protein (GFP). The modifier proteins are described in Supplementary Table . The CCR5 DNA was digested with the purified enzymes and resolved on an agarose gel. Successful digestion cuts the 1.5 kb substrate DNA into two 0.75 kb DNAs. ( C ) DNA band intensities in the gel image. Statistical comparison was made between C9 and each of the C9R or C9G proteins. Significance was indicated on top of each bar. One-way ANOVA using Tukey’s multiple-comparison test. The fold change relative to C9 is indicated above each bar. ( D ) DNA insertion and deletion (Indel) efficiency of C9, C9R, and C9G for the CCR5 gene in HEK293T cells at 8, 16, and 24 h after RNP transfection as measured by a T7E1 assay. Averages from the three replicate experiments are plotted. Two-way ANOVA using Tukey’s multiple-comparison test. ( E ) Indel efficiency of C9, C9R, and C9G for the CCR5 , HPRT1 , and EMX1 genes (left to right) in HEK293T cells. At 24 h ( CCR5 and HPRT1 ) and 8 h ( EMX1 ) post-transfection, cells were harvested and subjected to targeted deep sequencing. The experiments were performed in triplicate. NT group represent the NGS data from non-treated control. One-way ANOVA using Tukey’s multiple-comparison test was performed using C9 data as control. ( F ) Distribution of the DNA deletion sizes after treatment with C9, C9R, and C9G. The number of deletion events around the protospacer site in the CCR5 gene was counted according to the 15 bp deletion intervals from targeted deep sequencing data (n = 3) data. ( G ) Distribution of DNA insertion sizes after treatment with C9, C9R, and C9G in the CCR5 gene. The number of insertion events was counted according to the 1 bp insertion intervals from targeted deep sequencing data (n = 3) data. 6–10 and 11–61 bp insertion intervals were also shown to include events with smaller numbers. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, and n.s. indicates not significant.

    Techniques Used: Protein Purification, Comparison, Activity Assay, Purification, Agarose Gel Electrophoresis, Transfection, Sequencing, Control

    Related Articles

    Amplification:

    Article Title: Hijacking and recording intracellular RNAs in human cells using eukaryotic reprogrammed tracrRNAs
    Article Snippet: For NG-PE7 expression plasmid cloning, pCMV-PE7 (Addgene no. 214812) plasmid was digested with SacI (New England Biolabs, R3156S) and BamHI New England Biolabs, R3136S). .. To prepare the insert DNA fragments, the NG-Cas9 region and RT region were amplified from SpCas9-NG plasmid (Addgene no. 138566) and pCMV-PEmax (Addgene no. 174820). .. All insert DNA fragments were amplified with Q5 hotstart High-Fidelity DNA polymerase (New England Biolabs, M0493L).

    Plasmid Preparation:

    Article Title: Hijacking and recording intracellular RNAs in human cells using eukaryotic reprogrammed tracrRNAs
    Article Snippet: For NG-PE7 expression plasmid cloning, pCMV-PE7 (Addgene no. 214812) plasmid was digested with SacI (New England Biolabs, R3156S) and BamHI New England Biolabs, R3136S). .. To prepare the insert DNA fragments, the NG-Cas9 region and RT region were amplified from SpCas9-NG plasmid (Addgene no. 138566) and pCMV-PEmax (Addgene no. 174820). .. All insert DNA fragments were amplified with Q5 hotstart High-Fidelity DNA polymerase (New England Biolabs, M0493L).

    Article Title: Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated MUTYH variants
    Article Snippet: .. Base editor (BE) plasmids were generated by taking either AncBE4max-P2A-GFP (Addgene plasmid #112100) or ABE7.10max-P2A-GFP (Addgene plasmid #112101) and replacing the C-terminal 300 amino acids of Cas9n with that from SpCas9-NG (Addgene plasmid #117919) using USER (Uracil-Specific Excision Reagent) cloning [ ] to produce cytosine base editor (CBE) and adenine base editor (ABE) variants that recognize a relaxed PAM (protospacer adjacent motif) of NG (N = any DNA base; G = Guanine). .. The intact mCherry-P2A-EGFP reporter plasmids were generated with USER cloning following New England Biolabs (NEB) protocols, by replacing the ABE gene in the ABE-P2A-EGFP plasmid (Addgene plasmid #112101) with the mCherry gene from the pBAD-mCherry plasmid (Addgene plasmid #54630).

    Article Title: Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing.
    Article Snippet: .. The full length of SpCas9-NGwas PCR from Addgene (Addgene plasmid #138566). .. The full length of HiFi-Cas9 was PCR from Addgene (Addgene plasmid #140563).

    Article Title: Identification of Site in the UTY Gene as Safe Harbor Locus on the Y Chromosome of Pig
    Article Snippet: .. The UTY -specific single guide RNA (sgRNA) was designed using the online website https://cctop.cos.uni-heidelberg.de/ , accessed on 6 September 2023, where the oligonucleotides encoding the sgRNA are annealed and assembled into the linearized px459-SpCas9-NG vector (Addgene Plasmid #171370, HedgehogBio, Shanghai, China). .. The oligonucleotides encoding the sgRNA were denatured using a PCR machine with the following program: 37 °C for 5 min, followed by 95 °C for 30 min and, finally, incubated at room temperature (25 °C) for 1 h. Subsequently, the annealed oligonucleotides were ligated to the BbsI-digested px459 vector (HedgehogBio, Shanghai, China).

    Article Title: Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated MUTYH variants.
    Article Snippet: .. Base editor (BE) plasmids were generated by taking either AncBE4max-P2A-GFP (Addgene plasmid #112100) or ABE7.10max-P2A-GFP (Addgene plasmid #112101) and replacing the C-terminal 300 amino acids of Cas9n with that D ow nloaded from https://academ ic.oup.com /nar/article/53/6/gkaf037/8099200 by guest on 07 April 2025 from SpCas9-NG (Addgene plasmid #117919) using USER (Uracil-Specific Excision Reagent) cloning [ 58 ] to produce cytosine base editor (CBE) and adenine base editor (ABE) variants that recognize a relaxed PAM (protospacer adjacent motif) of NG (N = any DNA base; G = Guanine). .. The intact mCherry-P2A-EGFP reporter plasmids were generated with USER cloning following New England Biolabs (NEB) protocols, by replacing the ABE gene in the ABE-P2AEGFP plasmid (Addgene plasmid #112101) with the mCherry gene from the pBAD-mCherry plasmid (Addgene plasmid #54630).

    Article Title: Inhibitors of RNA-guided nuclease target binding and uses thereof
    Article Snippet: In the plasmid nucleofection method, cells were nucleofected either with 440 ng of SpCas9 (Addgene Plasmid #43861) or 400 ng of SpCas9 along with 40 ng of sgRNA (pFYF1320 eGFP Site #1, Addgene Plasmid #47511). .. In the plasmid nucleofection method, cells were nucleofected either with 440 ng of SpCas9 (Addgene Plasmid #43861) or 400 ng of SpCas9 along with 40 ng of sgRNA (pFYF1320 eGFP Site #1, Addgene Plasmid #47511). ..

    Generated:

    Article Title: Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated MUTYH variants
    Article Snippet: .. Base editor (BE) plasmids were generated by taking either AncBE4max-P2A-GFP (Addgene plasmid #112100) or ABE7.10max-P2A-GFP (Addgene plasmid #112101) and replacing the C-terminal 300 amino acids of Cas9n with that from SpCas9-NG (Addgene plasmid #117919) using USER (Uracil-Specific Excision Reagent) cloning [ ] to produce cytosine base editor (CBE) and adenine base editor (ABE) variants that recognize a relaxed PAM (protospacer adjacent motif) of NG (N = any DNA base; G = Guanine). .. The intact mCherry-P2A-EGFP reporter plasmids were generated with USER cloning following New England Biolabs (NEB) protocols, by replacing the ABE gene in the ABE-P2A-EGFP plasmid (Addgene plasmid #112101) with the mCherry gene from the pBAD-mCherry plasmid (Addgene plasmid #54630).

    Article Title: Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated MUTYH variants.
    Article Snippet: .. Base editor (BE) plasmids were generated by taking either AncBE4max-P2A-GFP (Addgene plasmid #112100) or ABE7.10max-P2A-GFP (Addgene plasmid #112101) and replacing the C-terminal 300 amino acids of Cas9n with that D ow nloaded from https://academ ic.oup.com /nar/article/53/6/gkaf037/8099200 by guest on 07 April 2025 from SpCas9-NG (Addgene plasmid #117919) using USER (Uracil-Specific Excision Reagent) cloning [ 58 ] to produce cytosine base editor (CBE) and adenine base editor (ABE) variants that recognize a relaxed PAM (protospacer adjacent motif) of NG (N = any DNA base; G = Guanine). .. The intact mCherry-P2A-EGFP reporter plasmids were generated with USER cloning following New England Biolabs (NEB) protocols, by replacing the ABE gene in the ABE-P2AEGFP plasmid (Addgene plasmid #112101) with the mCherry gene from the pBAD-mCherry plasmid (Addgene plasmid #54630).

    Cloning:

    Article Title: Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated MUTYH variants
    Article Snippet: .. Base editor (BE) plasmids were generated by taking either AncBE4max-P2A-GFP (Addgene plasmid #112100) or ABE7.10max-P2A-GFP (Addgene plasmid #112101) and replacing the C-terminal 300 amino acids of Cas9n with that from SpCas9-NG (Addgene plasmid #117919) using USER (Uracil-Specific Excision Reagent) cloning [ ] to produce cytosine base editor (CBE) and adenine base editor (ABE) variants that recognize a relaxed PAM (protospacer adjacent motif) of NG (N = any DNA base; G = Guanine). .. The intact mCherry-P2A-EGFP reporter plasmids were generated with USER cloning following New England Biolabs (NEB) protocols, by replacing the ABE gene in the ABE-P2A-EGFP plasmid (Addgene plasmid #112101) with the mCherry gene from the pBAD-mCherry plasmid (Addgene plasmid #54630).

    Article Title: Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated MUTYH variants.
    Article Snippet: .. Base editor (BE) plasmids were generated by taking either AncBE4max-P2A-GFP (Addgene plasmid #112100) or ABE7.10max-P2A-GFP (Addgene plasmid #112101) and replacing the C-terminal 300 amino acids of Cas9n with that D ow nloaded from https://academ ic.oup.com /nar/article/53/6/gkaf037/8099200 by guest on 07 April 2025 from SpCas9-NG (Addgene plasmid #117919) using USER (Uracil-Specific Excision Reagent) cloning [ 58 ] to produce cytosine base editor (CBE) and adenine base editor (ABE) variants that recognize a relaxed PAM (protospacer adjacent motif) of NG (N = any DNA base; G = Guanine). .. The intact mCherry-P2A-EGFP reporter plasmids were generated with USER cloning following New England Biolabs (NEB) protocols, by replacing the ABE gene in the ABE-P2AEGFP plasmid (Addgene plasmid #112101) with the mCherry gene from the pBAD-mCherry plasmid (Addgene plasmid #54630).

    Polymerase Chain Reaction:

    Article Title: Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing.
    Article Snippet: .. The full length of SpCas9-NGwas PCR from Addgene (Addgene plasmid #138566). .. The full length of HiFi-Cas9 was PCR from Addgene (Addgene plasmid #140563).



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    Marked increase in indel efficiency of the Cas9-RecJ and Cas9-GFP chimeric fusion proteins. ( A ) Schematic of a translational fusion of a modifier protein to <t>SpyCas9.</t> His tag (6X His) for protein purification and nuclear localization signal (NLS) were placed. ( B ) Comparison of the DNA cleavage activity of SpyCas9 and its chimeric proteins, C-terminal chimeric SpyCas9 translational fusion proteins with the nucleases RecJ, RecE, T5, lambda, mung bean exonuclease, and human terminal deoxynucleotidyl transferase (TdT), and with green fluorescent protein (GFP). The modifier proteins are described in Supplementary Table . The CCR5 DNA was digested with the purified enzymes and resolved on an agarose gel. Successful digestion cuts the 1.5 kb substrate DNA into two 0.75 kb DNAs. ( C ) DNA band intensities in the gel image. Statistical comparison was made between C9 and each of the C9R or C9G proteins. Significance was indicated on top of each bar. One-way ANOVA using Tukey’s multiple-comparison test. The fold change relative to C9 is indicated above each bar. ( D ) DNA insertion and deletion (Indel) efficiency of C9, C9R, and C9G for the CCR5 gene in HEK293T cells at 8, 16, and 24 h after RNP transfection as measured by a T7E1 assay. Averages from the three replicate experiments are plotted. Two-way ANOVA using Tukey’s multiple-comparison test. ( E ) Indel efficiency of C9, C9R, and C9G for the CCR5 , HPRT1 , and EMX1 genes (left to right) in HEK293T cells. At 24 h ( CCR5 and HPRT1 ) and 8 h ( EMX1 ) post-transfection, cells were harvested and subjected to targeted deep sequencing. The experiments were performed in triplicate. NT group represent the NGS data from non-treated control. One-way ANOVA using Tukey’s multiple-comparison test was performed using C9 data as control. ( F ) Distribution of the DNA deletion sizes after treatment with C9, C9R, and C9G. The number of deletion events around the protospacer site in the CCR5 gene was counted according to the 15 bp deletion intervals from targeted deep sequencing data (n = 3) data. ( G ) Distribution of DNA insertion sizes after treatment with C9, C9R, and C9G in the CCR5 gene. The number of insertion events was counted according to the 1 bp insertion intervals from targeted deep sequencing data (n = 3) data. 6–10 and 11–61 bp insertion intervals were also shown to include events with smaller numbers. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, and n.s. indicates not significant.
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    Image Search Results


    Marked increase in indel efficiency of the Cas9-RecJ and Cas9-GFP chimeric fusion proteins. ( A ) Schematic of a translational fusion of a modifier protein to SpyCas9. His tag (6X His) for protein purification and nuclear localization signal (NLS) were placed. ( B ) Comparison of the DNA cleavage activity of SpyCas9 and its chimeric proteins, C-terminal chimeric SpyCas9 translational fusion proteins with the nucleases RecJ, RecE, T5, lambda, mung bean exonuclease, and human terminal deoxynucleotidyl transferase (TdT), and with green fluorescent protein (GFP). The modifier proteins are described in Supplementary Table . The CCR5 DNA was digested with the purified enzymes and resolved on an agarose gel. Successful digestion cuts the 1.5 kb substrate DNA into two 0.75 kb DNAs. ( C ) DNA band intensities in the gel image. Statistical comparison was made between C9 and each of the C9R or C9G proteins. Significance was indicated on top of each bar. One-way ANOVA using Tukey’s multiple-comparison test. The fold change relative to C9 is indicated above each bar. ( D ) DNA insertion and deletion (Indel) efficiency of C9, C9R, and C9G for the CCR5 gene in HEK293T cells at 8, 16, and 24 h after RNP transfection as measured by a T7E1 assay. Averages from the three replicate experiments are plotted. Two-way ANOVA using Tukey’s multiple-comparison test. ( E ) Indel efficiency of C9, C9R, and C9G for the CCR5 , HPRT1 , and EMX1 genes (left to right) in HEK293T cells. At 24 h ( CCR5 and HPRT1 ) and 8 h ( EMX1 ) post-transfection, cells were harvested and subjected to targeted deep sequencing. The experiments were performed in triplicate. NT group represent the NGS data from non-treated control. One-way ANOVA using Tukey’s multiple-comparison test was performed using C9 data as control. ( F ) Distribution of the DNA deletion sizes after treatment with C9, C9R, and C9G. The number of deletion events around the protospacer site in the CCR5 gene was counted according to the 15 bp deletion intervals from targeted deep sequencing data (n = 3) data. ( G ) Distribution of DNA insertion sizes after treatment with C9, C9R, and C9G in the CCR5 gene. The number of insertion events was counted according to the 1 bp insertion intervals from targeted deep sequencing data (n = 3) data. 6–10 and 11–61 bp insertion intervals were also shown to include events with smaller numbers. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, and n.s. indicates not significant.

    Journal: Scientific Reports

    Article Title: Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins

    doi: 10.1038/s41598-021-95406-8

    Figure Lengend Snippet: Marked increase in indel efficiency of the Cas9-RecJ and Cas9-GFP chimeric fusion proteins. ( A ) Schematic of a translational fusion of a modifier protein to SpyCas9. His tag (6X His) for protein purification and nuclear localization signal (NLS) were placed. ( B ) Comparison of the DNA cleavage activity of SpyCas9 and its chimeric proteins, C-terminal chimeric SpyCas9 translational fusion proteins with the nucleases RecJ, RecE, T5, lambda, mung bean exonuclease, and human terminal deoxynucleotidyl transferase (TdT), and with green fluorescent protein (GFP). The modifier proteins are described in Supplementary Table . The CCR5 DNA was digested with the purified enzymes and resolved on an agarose gel. Successful digestion cuts the 1.5 kb substrate DNA into two 0.75 kb DNAs. ( C ) DNA band intensities in the gel image. Statistical comparison was made between C9 and each of the C9R or C9G proteins. Significance was indicated on top of each bar. One-way ANOVA using Tukey’s multiple-comparison test. The fold change relative to C9 is indicated above each bar. ( D ) DNA insertion and deletion (Indel) efficiency of C9, C9R, and C9G for the CCR5 gene in HEK293T cells at 8, 16, and 24 h after RNP transfection as measured by a T7E1 assay. Averages from the three replicate experiments are plotted. Two-way ANOVA using Tukey’s multiple-comparison test. ( E ) Indel efficiency of C9, C9R, and C9G for the CCR5 , HPRT1 , and EMX1 genes (left to right) in HEK293T cells. At 24 h ( CCR5 and HPRT1 ) and 8 h ( EMX1 ) post-transfection, cells were harvested and subjected to targeted deep sequencing. The experiments were performed in triplicate. NT group represent the NGS data from non-treated control. One-way ANOVA using Tukey’s multiple-comparison test was performed using C9 data as control. ( F ) Distribution of the DNA deletion sizes after treatment with C9, C9R, and C9G. The number of deletion events around the protospacer site in the CCR5 gene was counted according to the 15 bp deletion intervals from targeted deep sequencing data (n = 3) data. ( G ) Distribution of DNA insertion sizes after treatment with C9, C9R, and C9G in the CCR5 gene. The number of insertion events was counted according to the 1 bp insertion intervals from targeted deep sequencing data (n = 3) data. 6–10 and 11–61 bp insertion intervals were also shown to include events with smaller numbers. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, and n.s. indicates not significant.

    Article Snippet: The SpyCas9 coding sequence (Addgene, #138566) was ligated into a pET28a vector (Merck Biosciences, Darmstadt, Germany) that had been linearized by Nco I and Xho I double digestion.

    Techniques: Protein Purification, Comparison, Activity Assay, Purification, Agarose Gel Electrophoresis, Transfection, Sequencing, Control