| Description |
GenCRISPR™ Ultra eSpCas9-2NLS-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.
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| Selection Guide |
GenScript provides both wild-type and mutant Cas9 nucleases in GMP Grade* for selection based on your specific downstream applications. The GMP Grade Cas9 nucleases can be utilized as ancillary materials for Cell, Gene, and Tissue-Based Products and are manufactured under the standards listed below:- § USP <1043>. Ancillary materials for Cell, Gene and Tissue-engineered products.
- § ICH Q7 Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients
- § NMPA: Technical guidelines for pharmaceutical research and evaluation of immune cell therapy products
- § Guideline and procedure specified in Chinese Pharmacopeia.
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Nucleases
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Description
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GenCRISPR™ Ultra
NLS-Cas9-GMP (Cat. No. Z03623-GMP)
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Recombinant
Cas9 nuclease (wild type) of Streptococcus
pyogenes; ideal choice for most CRISPR gene editing applications where
high editing efficiency is preferred.
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GenCRISPR™ Ultra
eSpCas9-2NLS-GMP (Cat. No. Z03624-GMP)
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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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| Source |
Recombinant mutant
Cas9 with nuclear localization signal (NLS) at both N-terminal and C-terminal
expressed in E.coli
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| Species |
S. pyogenes
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| Tag |
Tag-free
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| Theoretical Molecular Weight |
~160 kDa
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| Concentration |
10.0 mg/ml
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| Active temperature |
Optimal at 37 °C
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| Application |
sgRNA-dependent double-stranded DNA cleavage. |
| Storage Buffer |
25 mM Tris-HCl, 300 mM NaCl, 0.1 mM EDTA, 50% Glycerol, pH 8.0
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| Storage & Stability |
Store at -20 ± 5°C for up to 12 months from the date of manufacture. Avoid repeated freeze-thaw cycles. |
| Appearance |
Clear, colorless, liquid
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| Identity |
Consistent with the theoretical sequence by LC-MS/MS |
| Purity |
≥ 95.0 as analyzed by SDS-PAGE
≥ 95.0 as analyzed by SEC-HPLC |
| Concentration by A280 |
10.0 mg/ml ± 1.0 mg/ml |
| Bioactivity (in vitro) |
≥ 85.0% |
| Residual DNase |
≤ 10 ng/mg
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| Residual RNase |
≤ 1 ng/mg
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| Endotoxin Level |
< 10 EU/mg
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| Residual HCP |
≤ 10 ng/mg
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| Residual HCD |
≤ 1 ng/mg
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| Mycoplasma |
Non-detectable by Culture method
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| Sterility |
No growth |
Figure 2: TRAC knock-out in primary T cells using GMP Grade GenCRISPR™ Ultra Cas9 or GenCRISPR™ Ultra eSpCas9 in different electroporation scale.
Figure 4: CIITA, GM-CSF and TGFBR2 knockout in Jurkat cells using GMP Grade GenCRISPR™ Ultra SpCas9 or GenCRISPR™ Ultra eSpCas9 Nuclease.
Figure 3: GFP gene knock-in at TRAC site in primary T cells using GMP Grade GenCRISPR™ Ultra Cas9 or GenCRISPR™ Ultra eSpCas9 Nuclease.
Figure 1: TRAC knock-out in primary T cells using GMP, basic GMP and RUO Grade GenCRISPR™ Ultra Cas9 or GenCRISPR™ Ultra eSpCas9 Nuclease.
| 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.
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| Annotation |
*GMP
Grade is a specific term that GenScript uses to describe the Cas9 nucleases
manufactured in GMP-complaint facility and in compliance with guidelines of
Current Good Manufacturing Practice (cGMP), ISO 9001 and ISO 13485 quality
management system standards with stringent process controls and complete
documentation records. GenScript is capable of providing documents, site
audits, and other support to help with the applications of your projects in
specific regions.
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For laboratory research or clinical research and manufacturing of
cell-based products. Direct human use, including taking orally and injection
are forbidden.
