目录产品 » GenCRISPR™/Cas9 基因编辑相关产品 » Cas9核酸酶 » GenCRISPR™ Ultra NLS-Cas9-basic GMP
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GenCRISPR™ Ultra NLS-Cas9-basic GMP

GenCRISPR™ Ultra NLS-Cas9-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 nuclear localization signal (NLS) expressed with the Cas9 nuclease, the RNP complex enters the nucleus and cleaves the 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.
询价
Z03623

产品概述
Description GenCRISPR™ Ultra NLS-Cas9-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 nuclear localization signal (NLS) expressed with the Cas9 nuclease, the RNP complex enters the nucleus and cleaves the 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.
Selection Guide GenScript provides two types of Cas9 nucleases in basic GMP* for selection based on your specific downstream applications.
NucleaseDescription
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. 
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应用指导
sgRNA-dependent double-stranded DNA cleavage.
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产品属性
Source Recombinant Cas9 nuclease (wt) with an N-terminal NLS 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 -20±5 °C for up to 12 months from the date of manufacture. Avoid repeated freeze-thaw cycles.
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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 ≥ 90.0%
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
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图例
GenCRISPR™ Ultra NLS-Cas9-basic GMP

Figure 1: TRAC and PD-1 knock-out efficiency in human primary T cells.
Human primary T cells were cultured for the test. 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 »

GenCRISPR™ Ultra NLS-Cas9-basic GMP

Figure 4: 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 »

GenCRISPR™ Ultra NLS-Cas9-basic GMP

Figure 3: RAB11A knock-out efficiency in 293T cells.
Human 293T cells were cultured for the test. The cells were transfected with GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) or wild type Cas9 proteins from competitors and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services) for human RAB11A gene knock-out by electroporation. After transfection and cell culture, the gene editing efficiency was analyzed by Sanger sequencing. GenScript GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) shows higher editing efficiency.
Note: This experiment used the following:
SpCas9: sgRNA (molar ratio) =1:3
RNP amount: 7.5 pmol
Cell number: 2.0 × 105 cells »

GenCRISPR™ Ultra NLS-Cas9-basic GMP

Figure 2: CD96 and NKG2A knock-out efficiency in NK92 cells.
Human NK92 cells were cultured for the test. The cells were transfected with GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) and sgRNA (synthesized by GenScript CRISPR Synthetic sgRNA Services) for human CD96 or NKG2A gene knock-out by electroporation. After transfection and cell culture, the gene editing efficiency were analyzed by Sanger sequencing. GenScript GenCRISPR™ Ultra NLS-SpCas9-basic GMP (Z03623) shows high editing efficiency.
Note: This experiment used the following:
SpCas9: sgRNA (molar ratio) = 1:1.2
RNP amount: 80 pmol
Cell number: 4.0 × 105 cells »

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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.

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