In vitro DNA cleavage assay with GenCrispr NLS-Cas9-EGFP nuclease
Reactions were set up according to recommended conditions, and cleavage products were resolved on a 1% agarose gel. Input DNA is EcoR V-linearized pUC57 plasmid DNA. Lane 1, DNA + gRNA; lane 2, marker; lane 3 and 4, DNA + gRNA + NLS-Cas9-EGFP 100 ng (lane 3) or 50 ng (lane 4).

Left: Cell transfection assay. 12 h after electroporation, cells were observed under bright or fluorescence microscope.
Right: in vivo gene editing efficiency assay by T7E1. Lane 1, marker; lane 2, negative control; lane 3, gRNA + NLS-Cas9-EGFP; lane 4, gRNA + NLS-Cas9.

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

GenCrispr NLS-Cas9-EGFP Nuclease

GenCrispr NLS-Cas9-EGFP is a fusion protein developed by GenScript. It contains a nuclear localization signal (NLS) on its N terminal end, and an EGFP and a 6x(His) sequence on the C terminal end. Cas9 nuclease is an RNA-guided endonuclease that can catalyze cleavage of double stranded DNA. This kind of targeted nuclease is a powerful tool for genome editing with high precision. Cas9 protein forms a highly stable ribonucleoprotein (RNP) complex with the guide RNA (gRNA) component of the CRISPR/Cas9 system. When Cas9 is expressed with an NLS sequence, the Cas9 RNP complex can localize to the nucleus immediately upon entering the cell. There is no requirement for in vivo transcription or translation, which improves the efficiency of this method over plasmid-based systems. Additionally, the Cas9 RNP complex is rapidly cleared from the cells minimizing the chance of off-target cleavage when compared to other systems (Kim, et al. 2014). The EGFP tag can be used as a reporter for tracking or sorting transfected cells, which enables enrichment of cell populations for desired genome edits via fluorescence activated cell sorting (FACS). It significantly reduces the labor and cost associated with single cell cloning and genotyping in genome editing applications.

Cat. No.	Z03393
Size	50 μg (1mg/ml) 100 μg (3mg/ml) Customized Size
Quantity
Price	¥34,075.00


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Description

_x000D_ GenCrispr NLS-Cas9-EGFP is a fusion protein developed by GenScript. It contains a nuclear localization signal (NLS) on its N terminal end, and an EGFP and a 6x(His) sequence on the C terminal end. Cas9 nuclease is an RNA-guided endonuclease that can catalyze cleavage of double stranded DNA. This kind of targeted nuclease is a powerful tool for genome editing with high precision. Cas9 protein forms a highly stable ribonucleoprotein (RNP) complex with the guide RNA (gRNA) component of the CRISPR/Cas9 system. When Cas9 is expressed with an NLS sequence, the Cas9 RNP complex can localize to the nucleus immediately upon entering the cell. There is no requirement for in vivo transcription or translation, which improves the efficiency of this method over plasmid-based systems. Additionally, the Cas9 RNP complex is rapidly cleared from the cells minimizing the chance of off-target cleavage when compared to other systems (Kim, et al. 2014). The EGFP tag can be used as a reporter for tracking or sorting transfected cells, which enables enrichment of cell populations for desired genome edits via fluorescence activated cell sorting (FACS). It significantly reduces the labor and cost associated with single cell cloning and genotyping in genome editing applications. _x000D_ _x000D_

_x000D_ Product Source: GenCrispr NLS-Cas9-EGFP is produced by expression from an E. coli strain. _x000D_
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_x000D_ Key Features_x000D_
_x000D_ - DNA-free: no external DNA added to system
_x000D_ - High cleavage efficiency: NLS ensures the efficient entry of the Cas9 protein into nuclei
_x000D_ - Low off target effects: transient expression of Cas9 nuclease improves specificity of cleavage
_x000D_ - Time-saving: no need for transcription and translation
_x000D_ - Reduced labor: enrich cell populations for desired genome edits via EGFP-based FACS. The C-terminal His-tag increases the choice of detection methods for the fusion protein.
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_x000D_ Applications
_x000D_ _x000D_ - Screening for highly efficient and specific targeting gRNAs by in vitro DNA cleavage.
_x000D_ - In vivo gene editing when combined with a specific gRNA by electroporation or injection.
_x000D_ - Enrichment of cell populations for desired genome edits via EGFP-based FACS.
_x000D_

Note

1000 nM is equal to 190 ng/µl.

Properties

Storage & Stability

GenCrispr NLS-Cas9-EGFP is supplied with 1X storage buffer (10 mM Tris, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% Glycerol pH7.4 at 25 °C) and recommended to be stored at -20 °C.

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