Purified Cas9 Protein (NLS-Cas9-NLS)

Maximize CRISPR/Cas9 performance and safety with transfectable, electroporatable, and injectable Cas9 Protein.
  • Highly concentrated and ready to use—no reconstitution required
  • Acts immediately upon entry into the cell
  • Rapid clearance reduces chances for off-target cleavage events
  • Avoids issues with genomic integration
  • Enables in-vitro cleavage assays

Products

Catalog Number Description Size Price Quantity Add to Cart
CAS410A-1 Purified Cas9 Protein (NLS-Cas9-NLS) 50 µg $150.00
- +
Contact Us

Overview

Overview

Take the power of CRISPR/Cas9 technology to the next level Highly concentrated and ready to use, Purified Cas9 Protein (NLS-Cas9-NLS) is ready for introduction into target cells via transfection, electroporation, or microinjection. Once inside the cell, two nuclear localization signals (NLS) working in concert direct the protein to the nucleus where it catalyzes gRNA-directed cleavage before being rapidly cleared. NLS-Cas9-NLS protein enables efficient genome editing while reducing off-target events1–3. By removing plasmid delivery of Cas9 from the genome editing process, both off-target events from random plasmid integration and the potential for an immune response from bacterial plasmid sequences are avoided1,2. In addition, the more transient nature of Cas9 protein compared to plasmid or mRNA delivery further reduces off-target activity without decreasing on-target efficiency1,2. The end result is better, safer Cas9 activity for applications where off-target events need to be minimized, such as:
  • Genome engineering in embryos
  • Disease model generation of organisms and cell lines
  • In vitro transfection of cells
  • In vitro cleavage assays for functional gRNA screens
With transfectable/electroporatable/injectable Cas9 protein, you can:
  • Increase Cas9 efficiency2
  • Reduce off-target events2
  • Reduce the potential for immune response to vector DNA
  • Simplify delivery to cells and embryos
  • Perform multiplex, high-throughput studies
  • Conduct typical downstream functional assays
Get the full system Streamline genome editing with transfectable/electroporatable Cas9 protein—get Cas9 protein bundled into a kit with the Cas9 Protein & T7 gRNA SmartNuclease™ Synthesis Kit (Cat.# CAS410A-KIT) . Not sure whether you need a CRISPR/Cas9 plasmid, purified protein, or mRNA? Use this table to choose the CRISPR/Cas9 product that’s right for you:
For This Application
In these types of cells
Use These Products
MODIFYING ORGANISMS

  • Gene tagging

  • Transgenic organism generation

  • Model organism engineering

Embryos—to create transgenic animalsInjectable Cas9 mRNA & gRNA Synthesis Kits
Cas9 Protein
EGFP-labeled Cas9 Protein
Animals models—in vivo genome editingAAV-Cas9 Vectors  
Cas9 Protein
EGFP-labeled Cas9 Protein
MODIFYING CELL LINES

  • Stable KO, KI, and genome editing of
    somatic cells

  • Transgenic cell line generation

  • Cell-based disease models

Cells that are transfectableCas9 Plasmids
Cas9 Protein
EGFP-labeled Cas9 Protein
Difficult-to-transfect cell lines:

  • Primary cells

  • Hematopoietic cells

  • Stem cells

AAV-Cas9 Vectors 
Lenti Cas9 Systems
SCREENING

  • Genome-wide surveys

  • gRNA library screens

  • Functional screens

All cell types requiring stable Cas9 overexpressionLenti Cas9 Systems
AAVS1 Safe Harbor Site Cas9
Gene Knock-in System
Cas9 Protein
EGFP-labeled Cas9 Protein
PRE-CLINICAL APPLICATIONS
  • Off-target events are of highest concern
All cell types and applicationsCas9 Nickase, available in all delivery formats
Cas9 Protein
EGFP-labeled Cas9 Protein
SIMULTANEOUS ENGINEERING OF MULTIPLE MUTATIONSAll cell types and applicationsMultiplex gRNA cloning kit, compatible with all Cas9 delivery options
References
  1. Ramakrishna, S. et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. (2014). 24:1020–1027. PMCID: PMC4032848.
  2. Wang, L. et al. Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos. Sci. Rep. (2015). 5:17517. PMCID: PMC4664917.
  3. Chen, S., et al. Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes. J. Biol. Chem. (2016). 291(28):14457-67. PMID: 27151215.

How It Works

How It Works

Using Transfectable/Electroporatable Cas9 Protein Using SBI’s transfectable or electroporatable Cas9 protein is quick and easy. Simply pre-incubate Cas9 protein with your gRNA and then either transfect or electroporate as normal. While a 1:1 ratio of Cas9 protein to gRNA was used for the study in the Supporting Data section below, we recommend optimizing the amounts and ratios for your specific gRNA and cell lines. For more general guidance on using CRISPR/Cas9 technology for genome engineering, take a look at our CRISPR/Cas9 tutorials as well as the following application notes: CRISPR/Cas9 Gene Knock-Out Application Note (PDF) » CRISPR/Cas9 Gene Editing Application Note (PDF) » CRISPR/Cas9 Gene Tagging Application Note (PDF) » CRISPR/Cas9 Basics Through careful selection of the target sequence and design of a donor plasmid for homologous recombination, you can achieve efficient and highly targeted genomic modification with CRISPR/Cas9. The system A quick overview of the CRISPR/Cas9 System.
Figure 1. The Cas9 system.
Cas9 protein—uses guide RNA (gRNA) to direct site-specific, double-strand DNA cleavage adjacent to a protospacer adapter motif (PAM) in the target DNA. gRNA—RNA sequence that guides Cas9 to cleave a homologous region in the target genome. Efficient cleavage only where the gRNA homology is adjacent to a PAM. PAM—protospacer adapter motif, NGG, is a target DNA sequence that spCas9 will cut upstream from if directed to by the gRNA. The workflow at-a-glance DESIGN: Select gRNA and HR donor plasmids. Choice of gRNA site and design of donor plasmid determines whether the homologous recombination event results in a knock-out, knock-in, edit, or tagging. CONSTRUCT: Clone gRNA into all-in-one Cas9 vector. Clone 5’ and 3’ homology arms into HR donor plasmid. If creating a knock-in, clone desired gene into HR donor. CO-TRANSFECT or CO-INJECT: Introduce Cas9, gRNA, and HR Donors into the target cells using co-transfection for plasmids, co-transduction for lentivirus, or co-injection for mRNAs. SELECT/SCREEN: Select or screen for mutants and verify. VALIDATE: Genotype or sequence putative mutants to verify single or biallelic conversion.

Supporting Data

Supporting Data

NLS-Cas9-NLS protein is functional in an in vivo assay as seen in a DNA gel To demonstrate that the NLS-Cas9-NLS protein possesses similar levels of activity as Cas9 introduced using a lentivector—our Cas9 All-in-one System—we created a gRNA that generates a one nt change in the endogenous miR-21 gene, delivered NLS-Cas9-EGFP using either CRISPRMax or cell-penetrating peptide (CPP), and performed a T7E1 mismatch detection assay (Figure 2). Levels of the cleavage product in cells with NLS-Cas9-NLS are similar to the levels generated using our Cas9 All-in-one System, demonstrating the activity of the protein.
Figure 2. NLS-Cas9-NLS protein is functional in an in vivo assay as seen in a DNA gel.

Resources

Citations

Products

Catalog Number Description Size Price Quantity Add to Cart
CAS410A-1 Purified Cas9 Protein (NLS-Cas9-NLS) 50 µg $150.00
- +
Contact Us

Overview

Overview

Take the power of CRISPR/Cas9 technology to the next level Highly concentrated and ready to use, Purified Cas9 Protein (NLS-Cas9-NLS) is ready for introduction into target cells via transfection, electroporation, or microinjection. Once inside the cell, two nuclear localization signals (NLS) working in concert direct the protein to the nucleus where it catalyzes gRNA-directed cleavage before being rapidly cleared. NLS-Cas9-NLS protein enables efficient genome editing while reducing off-target events1–3. By removing plasmid delivery of Cas9 from the genome editing process, both off-target events from random plasmid integration and the potential for an immune response from bacterial plasmid sequences are avoided1,2. In addition, the more transient nature of Cas9 protein compared to plasmid or mRNA delivery further reduces off-target activity without decreasing on-target efficiency1,2. The end result is better, safer Cas9 activity for applications where off-target events need to be minimized, such as:
  • Genome engineering in embryos
  • Disease model generation of organisms and cell lines
  • In vitro transfection of cells
  • In vitro cleavage assays for functional gRNA screens
With transfectable/electroporatable/injectable Cas9 protein, you can:
  • Increase Cas9 efficiency2
  • Reduce off-target events2
  • Reduce the potential for immune response to vector DNA
  • Simplify delivery to cells and embryos
  • Perform multiplex, high-throughput studies
  • Conduct typical downstream functional assays
Get the full system Streamline genome editing with transfectable/electroporatable Cas9 protein—get Cas9 protein bundled into a kit with the Cas9 Protein & T7 gRNA SmartNuclease™ Synthesis Kit (Cat.# CAS410A-KIT) . Not sure whether you need a CRISPR/Cas9 plasmid, purified protein, or mRNA? Use this table to choose the CRISPR/Cas9 product that’s right for you:
For This Application
In these types of cells
Use These Products
MODIFYING ORGANISMS

  • Gene tagging

  • Transgenic organism generation

  • Model organism engineering

Embryos—to create transgenic animalsInjectable Cas9 mRNA & gRNA Synthesis Kits
Cas9 Protein
EGFP-labeled Cas9 Protein
Animals models—in vivo genome editingAAV-Cas9 Vectors  
Cas9 Protein
EGFP-labeled Cas9 Protein
MODIFYING CELL LINES

  • Stable KO, KI, and genome editing of
    somatic cells

  • Transgenic cell line generation

  • Cell-based disease models

Cells that are transfectableCas9 Plasmids
Cas9 Protein
EGFP-labeled Cas9 Protein
Difficult-to-transfect cell lines:

  • Primary cells

  • Hematopoietic cells

  • Stem cells

AAV-Cas9 Vectors 
Lenti Cas9 Systems
SCREENING

  • Genome-wide surveys

  • gRNA library screens

  • Functional screens

All cell types requiring stable Cas9 overexpressionLenti Cas9 Systems
AAVS1 Safe Harbor Site Cas9
Gene Knock-in System
Cas9 Protein
EGFP-labeled Cas9 Protein
PRE-CLINICAL APPLICATIONS
  • Off-target events are of highest concern
All cell types and applicationsCas9 Nickase, available in all delivery formats
Cas9 Protein
EGFP-labeled Cas9 Protein
SIMULTANEOUS ENGINEERING OF MULTIPLE MUTATIONSAll cell types and applicationsMultiplex gRNA cloning kit, compatible with all Cas9 delivery options
References
  1. Ramakrishna, S. et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. (2014). 24:1020–1027. PMCID: PMC4032848.
  2. Wang, L. et al. Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos. Sci. Rep. (2015). 5:17517. PMCID: PMC4664917.
  3. Chen, S., et al. Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes. J. Biol. Chem. (2016). 291(28):14457-67. PMID: 27151215.

How It Works

How It Works

Using Transfectable/Electroporatable Cas9 Protein Using SBI’s transfectable or electroporatable Cas9 protein is quick and easy. Simply pre-incubate Cas9 protein with your gRNA and then either transfect or electroporate as normal. While a 1:1 ratio of Cas9 protein to gRNA was used for the study in the Supporting Data section below, we recommend optimizing the amounts and ratios for your specific gRNA and cell lines. For more general guidance on using CRISPR/Cas9 technology for genome engineering, take a look at our CRISPR/Cas9 tutorials as well as the following application notes: CRISPR/Cas9 Gene Knock-Out Application Note (PDF) » CRISPR/Cas9 Gene Editing Application Note (PDF) » CRISPR/Cas9 Gene Tagging Application Note (PDF) » CRISPR/Cas9 Basics Through careful selection of the target sequence and design of a donor plasmid for homologous recombination, you can achieve efficient and highly targeted genomic modification with CRISPR/Cas9. The system A quick overview of the CRISPR/Cas9 System.
Figure 1. The Cas9 system.
Cas9 protein—uses guide RNA (gRNA) to direct site-specific, double-strand DNA cleavage adjacent to a protospacer adapter motif (PAM) in the target DNA. gRNA—RNA sequence that guides Cas9 to cleave a homologous region in the target genome. Efficient cleavage only where the gRNA homology is adjacent to a PAM. PAM—protospacer adapter motif, NGG, is a target DNA sequence that spCas9 will cut upstream from if directed to by the gRNA. The workflow at-a-glance DESIGN: Select gRNA and HR donor plasmids. Choice of gRNA site and design of donor plasmid determines whether the homologous recombination event results in a knock-out, knock-in, edit, or tagging. CONSTRUCT: Clone gRNA into all-in-one Cas9 vector. Clone 5’ and 3’ homology arms into HR donor plasmid. If creating a knock-in, clone desired gene into HR donor. CO-TRANSFECT or CO-INJECT: Introduce Cas9, gRNA, and HR Donors into the target cells using co-transfection for plasmids, co-transduction for lentivirus, or co-injection for mRNAs. SELECT/SCREEN: Select or screen for mutants and verify. VALIDATE: Genotype or sequence putative mutants to verify single or biallelic conversion.

Supporting Data

Supporting Data

NLS-Cas9-NLS protein is functional in an in vivo assay as seen in a DNA gel To demonstrate that the NLS-Cas9-NLS protein possesses similar levels of activity as Cas9 introduced using a lentivector—our Cas9 All-in-one System—we created a gRNA that generates a one nt change in the endogenous miR-21 gene, delivered NLS-Cas9-EGFP using either CRISPRMax or cell-penetrating peptide (CPP), and performed a T7E1 mismatch detection assay (Figure 2). Levels of the cleavage product in cells with NLS-Cas9-NLS are similar to the levels generated using our Cas9 All-in-one System, demonstrating the activity of the protein.
Figure 2. NLS-Cas9-NLS protein is functional in an in vivo assay as seen in a DNA gel.

Citations