GenCRISPR™ ゲノム編集関連製品 » GenCRISPR™ ヌクレアーゼ-CRISPR遺伝子編集 » GenCRISPR™ Cas9 v1.2
GenCRISPR™ Cas9 V1.2

A 20 μl reaction in 1 × Cas9 Nuclease Reaction Buffer containing linearized plasmid, gRNA, and Cas9 for 1 hour at 37 °C results in a digestion efficiency of linearized plasmid higher than 90%, as determined by agarose gel electrophoresis.

GenCRISPR™ Cas9 V1.2

Human Jurkat cells were cultured for the test. The cells were transfected with GenCRISPR™ Cas9 v1.2 (Z03702)+ sgRNA (synthesized from GenScript gene@genscript.com) for human TCRαβ gene knock out by electroporation. After transfection and cell culture, measure the gene editing efficiency. GenCRISPR™ Cas9 v1.2 shows a much high editing efficiency.

GenCRISPR™ Cas9 V1.2

Human T cells were cultured for the test. The cells were transfected with GenCRISPR™ Cas9 v1.2 (Z03702)+ sgRNA (synthesized from GenScript gene@genscript.com) + dsDNA template (synthesized from GenScript gene@genscript.com) for knock in test at TCRαβ site by electroporation. After transfection and cell culture, measure the editing efficiency.

GenCRISPR™ Cas9 v1.2

GenCRISPR™ Cas9 v1.2 is a tag free nuclease produced by expression in an E. coli strain carrying a plasmid encoding the Cas9 gene from Streptococcus pyogenes with a biparticle nucleus localization signal (BPNLS) at N-terminal and a nucleoplasmin nucleus localization signal (nucleoplasmin NLS) at C-terminal. It has been reported that BPNLS and nucleoplasmin NLS are able to improve the gene editing efficiency.
Z03702
¥34,075.00

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Description

The GenCRISPR™_x000D_ Cas9 v1.2 can be formed_x000D_ with the guide RNA into a ribonucleoprotien (RNP) complex. The use of an RNP_x000D_ complex to perform gene editing has been shown to reduce the challenges_x000D_ encountered with other CRISPR gene editing techniques such as viral and plasmid_x000D_ delivery. Challenges include off-target effects, cell viability and transcription/translational_x000D_ challenges.

_x000D_ _x000D_ _x000D_ _x000D_ GenCRISPR™ Cas9 v1.2 is a tag_x000D_ free nuclease produced by expression in an E._x000D_ coli strain carrying a plasmid encoding the Cas9 gene from Streptococcus pyogenes with a biparticle_x000D_ nucleus localization signal (BPNLS) at N-terminal and a nucleoplasmin nucleus_x000D_ localization signal (nucleoplasmin NLS) at C-terminal. It has been reported_x000D_ that BPNLS and nucleoplasmin NLS are able to improve the gene editing_x000D_ efficiency. 
Note

For laboratory research use only. Direct human use, including taking orally and injection and clinical use are forbidden.

Applications
gRNA-dependent double-stranded DNA cleavage

Properties
Source Recombinant Cas9 with a BPNLS at N-terminal and a nucleoplasmin NLS at C-terminal expressed by E.coli 
Species Streptococcus pyogenes
Tag Tag-free
Molecular Weight ~160 kDa
Concentration 4 mg/ml
Active temperature This Cas9 is active at 37°C.
Formulation Supplied as a solution of 25 mM Tris, 300 mM NaCl, 0.1 mM EDTA, 50% glycerol, pH 8.0.
Storage & Stability This product remains stable for up to 12 months at -20°C. Avoid repeated freeze-thaw cycles.
Key Features High gene editing efficiencies: Consistent high editing efficiency in in-vitro and in-vivo.
Tag-free: Amino acid is free from additional tagging amino acid. 
DNA-free: No external DNA added to the system.

Quality Control Specifications
Appearance Clear, colorless liquid
Purity ≥ 90% as analyzed by SDS-PAGE
Concentration by A280 4 mg/ml±10% 
Bioactivity (in vitro) ≥ 90%
Residual DNase Non-specific DNase activity
Residual RNase Non-specific RNase activity
Endotoxin Level ≤ 100 EU/mg as analyzed by gel clotting method

Examples
  • GenCRISPR™ Cas9 V1.2
    • GenCRISPR™ Cas9 V1.2

    A 20 μl reaction in 1 × Cas9 Nuclease Reaction Buffer containing linearized plasmid, gRNA, and Cas9 for 1 hour at 37 °C results in a digestion efficiency of linearized plasmid higher than 90%, as determined by agarose gel electrophoresis.

  • GenCRISPR™ Cas9 V1.2
    • GenCRISPR™ Cas9 V1.2

    Human Jurkat cells were cultured for the test. The cells were transfected with GenCRISPR™ Cas9 v1.2 (Z03702)+ sgRNA (synthesized from GenScript gene@genscript.com) for human TCRαβ gene knock out by electroporation. After transfection and cell culture, measure the gene editing efficiency. GenCRISPR™ Cas9 v1.2 shows a much high editing efficiency.

  • GenCRISPR™ Cas9 V1.2
    • GenCRISPR™ Cas9 V1.2

    Human T cells were cultured for the test. The cells were transfected with GenCRISPR™ Cas9 v1.2 (Z03702)+ sgRNA (synthesized from GenScript gene@genscript.com) + dsDNA template (synthesized from GenScript gene@genscript.com) for knock in test at TCRαβ site by electroporation. After transfection and cell culture, measure the editing efficiency.


Background
References 1.      Liu, Xinyi, et al. "Improving editing efficiency for the sequences with NGH PAM using xCas9-derived base editors." Molecular Therapy-Nucleic Acids 17 (2019): 626-635. 
2.      Pollard, Victoria W., et al. "A novel receptor-mediated nuclear protein import pathway." Cell 86.6 (1996): 985-994. 
3.      Koblan, Luke W., et al. "Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction." Nature biotechnology 36.9 (2018): 843-846.