GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP

GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP is utilized for CRISPR gene editing applications. The Cas9 nuclease forms a stable ribonucleoprotein (RNP) complex with the guide RNA (gRNA) component. With the help of two nuclear localization signals (NLS) expressed with the Cas9 nuclease, the RNP complex enters the nucleus and cleaves target gene. When compared with a plasmid-based delivery system, the RNP delivery system has been observed to increase the on-target gene editing efficiency and decrease off-target effects.

价格	询价
产品编号	Z03624
规格	100 μg 500 μg 1 mg 大单询价
数量

产品概述应用指导产品属性质控参数图例技术背景

产品概述

Description

Selection Guide

GenScript provides two types of Cas9 nucleases in basic GMP* for selection based on your specific downstream applications.

Nucleases	Description
GenCRISPR™ Ultra NLS-Cas9-basic GMP (Z03623)	Recombinant Cas9 nuclease (wild type) of Streptococcus pyogenes; ideal choice for most CRISPR gene editing applications where high editing efficiency is preferred.
GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624)	Recombinant enhanced specificity Cas9 nuclease (mutant) of Streptococcus pyogenes; ideal for CRISPR gene editing applications which require low off-target effects and robust on-target cleavage.

应用指导

sgRNA-dependent double-stranded DNA cleavage.

产品属性

Source	Recombinant mutant Cas9 with nuclear localization signal (NLS) at both N-terminal and C-terminal expressed in E.coli
Species	S. pyogenes
Tag	Tag-free
Theoretical Molecular Weight	~160 kDa
Concentration	10.0 mg/ml
Active temperature	optimal at 37 °C.
Storage Buffer	25 mM Tris-HCl, 300 mM NaCl, 0.1 mM EDTA, 50% Glycerol, pH 8.0.
Storage & Stability	Store at -80±10 °C for up to 12 months from the date of manufacture. Avoid freeze-thaw more than 5 times.

质控参数

Appearance	Clear, colorless, liquid
Purity	≥ 95.0% as analyzed by SDS-PAGE ≥ 95.0% as analyzed by SEC-HPLC
Concentration	10.0 mg/ml ± 1.0 mg/ml
Bioactivity	≥ 85.0%
Residual DNase	≤ 10 ng/mg
Residual RNase	≤ 1 ng/mg
Endotoxin Level	< 10 EU/mg
Residual HCP	≤ 10 ng/mg
Residual HCD	≤ 1 ng/mg
Bioburden	< 1 CFU/ml
Mycoplasma	< LOD

图例

Figure 5: Gene knock-in efficiency at TRAC site in primary T cells.
The cells were transfected with GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) or GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624), sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services) and dsDNA HDR template (synthesized by GenScript Double-strand DNA Synthesis Services) for gene knock-in at TRAC site in primary T cells by electroporation. After transfection and cell culture, the gene knock-in efficiency was analyzed by FACS. Both GenScript GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) and GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) show high gene knock-in efficiency.
Note: This experiment used the following:
SpCas9/eSpCas9: sgRNA (molar ratio) =1:2
RNP amount: ~ 25 pmol
Cell number: 1.0 × 10⁶ cells »

Figure 4. TRAC knock-out efficiency in 293 T cells.
The cells were transfected with GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) or GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) or wild type Cas9 from competitor and same sgRNA for human TRAC gene knock-out by electroporation. After transfection and cell culture, the gene editing efficiency were measured by Sanger sequencing. Data shows that GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) demonstrate a lower gene editing efficiency with a small amount of RNP than wild type SpCas9 from both GenScript and competitor, while increasing a little RNP amount exhibits a comparable editing efficiency with wild type SpCas9.
Note: This experiment used the following:
Cas9: sgRNA (molar ratio) =1:3
RNP amount: 1-7.5 pmol
Cell number: 2.0 × 10⁵ cells »

Figure 3: TRAC knock-out and off-target effect analysis in 293 T cells.
The cells were transfected with GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) or wild type Cas9 proteins from competitors and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services) for human TRAC gene knock-out by electroporation. After transfection and cell culture, the TRAC knock-out efficiency and off-target effect were analyzed by Sanger sequencing. GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) shows a comparative gene editing efficiency to competing products (wild type SpCas9), but a greatly lower off-target effect.
Note: This experiment used the following:
eSpCas9: sgRNA (molar ratio) =1:3
RNP amount: 5-7.5 pmol
Cell number: 2.0 × 10⁵ cells »

Figure 2: NKG2A knock-out efficiency in NK92 cells.
The cells were transfected with GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) or GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services) for human NKG2A gene knock-out by electroporation. After transfection and cell culture, the NKG2A knock-out efficiency was analyzed by Sanger sequencing. Both GenScript GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) or GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) show high gene editing efficiency.
Note: This experiment used the following:
SpCas9/eSpCas9: sgRNA (molar ratio) = 1:1.2
RNP amount: 80 pmol
Cell number: 4.0 × 10⁵ cells »

Figure 1: TRAC and PD-1 knock-out efficiency in human primary T cells.
The cells were transfected with GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) or GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP (Z03624) or wild type Cas9 and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services ) for human TRAC and PD-1 gene knock-out by electroporation. After transfection and cell culture, the TRAC knock-out efficiency was measured by FACS while PD-1 knock-out efficiency was measured by Sanger sequencing. Both GenScript Ultra SpCas9 and eSpCas9 show high editing efficiency.
Note: This experiment used the following:
Cas9: sgRNA (molar ratio) =1:2
RNP amount: ~ 25 pmol
Cell number: 1.0 × 10⁶ cells »

A 20 μl reaction in 1 × Cas9 Nuclease Reaction Buffer containing linearized plasmid, gRNA, and eSpCas9-N-NLS for 2 hour at 37 °C results in a digestion efficiency of linearized plasmid higher than 90%, as determined by agarose gel electrophoresis. »

技术背景

References

1. Jinek, Martin, et al. "A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity." science 337.6096 (2012): 816-821.
2. Chen, Sean, et al. "Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes." Journal of Biological Chemistry 291.28 (2016): 14457-14467.
3. Larson, Matthew H., et al. "CRISPR interference (CRISPRi) for sequence-specific control of gene expression." Nature protocols 8.11 (2013): 2180-2196.
4. Ran, F. Ann, et al. "Genome engineering using the CRISPR-Cas9 system." Nature protocols 8.11 (2013): 2281-2308.
5. Kim, Sojung, et al. "Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins." Genome research 24.6 (2014): 1012-1019.

Annotation

*basic GMP is a branding term that GenScript uses to describe reagents manufactured in compliance with ISO 9001 and ISO 13485 quality management system standards and with more stringent process controls and relatively complete documentation records. GenScript is capable of providing documents, site audit and other support to help with the applications of your projects in specific regions.

For research or manufacturing use. Not intended for clinical use. Direct human use, including taking orally and injection are forbidden.

GenCRISPR™ Ultra eSpCas9-2NLS-basic GMP

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