Cas9 Protein & T7 gRNA SmartNuclease™ Synthesis Kit

Streamline your genome editing workflow with purified Cas9 protein (NLS-Cas9-NLS or NLS-Cas9-EGFP) bundled with the T7 gRNA Cloning and Production Kit.

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

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Products

Catalog Number	Description	Size	Price	Quantity	Add to Cart
CAS420A-KIT	EGFP-labeled Cas9 Protein (NLS-Cas9-EGFP) and T7 gRNA SmartNuclease Synthesis Kit	1 Kit (10 Reactions)	$859	Contact Us			- +
CAS410A-KIT	Purified Cas9 Protein (NLS-Cas9-NLS) and T7 gRNA SmartNuclease Synthesis Kit	1 Kit (10 Reactions)	$828	Contact Us			- +

Overview

Get Purified Cas9 Protein bundled with gRNA production

Combining a T7 gRNA SmartNuclease™ Synthesis Kit with purified, transfection-/electroporation-ready Cas9 Protein or EGFP-labeled Cas9 Protein gives you everything you need for targeted genome editing (note that many applications including gene knock-outs, knock-ins, and edits also require the use of a homologous recombination (HR) targeting vector—see our CRISPR/Cas9 tutorials to learn more).

Cas9 Protein & T7 gRNA SmartNuclease™ Synthesis Kit includes Purified Cas9 Protein (NLS-Cas9-NLS, Cat.# CAS410A-1) or EGFP-labeled Cas 9 Protein (NLS-Cas9-EGFP, Cat.# CAS420A-1) and the T7 gRNA SmartNuclease Synthesis Kit (Cat.# CAS510A-KIT).

Taking CRISPR/Cas9 technology to the next level

With the Cas9 protein, you can get more efficient genome editing while reducing off-target events^1–3. 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 avoided^1,2. In addition, the more transient nature of Cas9 protein compared to plasmid or mRNA delivery further reduces off-target activity without decreasing on-target efficiency^1,2. 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 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

Efficiently generate gRNA with the T7 gRNA SmartNuclease Synthesis Kit

The T7 gRNA SmartNuclease Synthesis Kit comes with a pre-linearized, ready-for-cloning gRNA expression vector (Figure 1) and all the reagents you need for T7-driven in vitro transcription of your cloned gRNA.

Figure 1. The T7 gRNA SmartNuclease Cloning and Production Vector.

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 animals	Injectable Cas9 mRNA & gRNA Synthesis Kits Cas9 Protein EGFP-labeled Cas9 Protein
	Animals models—in vivo genome editing	AAV-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 transfectable	Cas9 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 overexpression	Lenti 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 applications	Cas9 Nickase, available in all delivery formats Cas9 Protein EGFP-labeled Cas9 Protein

SIMULTANEOUS ENGINEERING OF MULTIPLE MUTATIONS	All cell types and applications	Multiplex gRNA cloning kit, compatible with all Cas9 delivery options

References

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.
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.
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.

References

How It Works

Using Transfectable/Electroporatable Cas9 Protein

Using SBI’s transfectable or electroporatable Cas9 protein/EGFP-labeled 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 2. 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

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 3). 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 3. NLS-Cas9-NLS protein is functional in an in vivo assay as seen in a DNA gel.

NLS-Cas9-EGFP can be transfected and electroporated into cells

NLS-Cas9-EGFP can be introduced into cells by transfection—using CRISPRMAX and a cell-penetrating peptide system (CPP)—as well by electroporation (Figure 4).

Figure 4. NLS-Cas9-EGFP can be transfected and electroporated into cells

NLS-Cas9-EGFP protein is functional in an in vitro assay

NLS-Cas9-EGFP can be used for in vitro studies (Figure 5). We linearized pUC57 plasmid DNA with EcoRV and assessed nuclease activity and specificity of NLS-Cas9-EGFP by appearance of a cleavage product. Lanes with gRNA and NLS-Cas9-EGFP exhibit the expected cleavage products in a dosage-dependent manner, demonstrating the in vitro activity and specificity of NLS-Cas9-EGFP.

Figure 5. NLS-Cas9-EGFP protein is functional in an in vitro assay.

NLS-Cas9-EGFP protein is functional in an in vivo assay

To demonstrate that the NLS-Cas9-EGFP 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 6A). Both methods of introducing NLS-Cas9-EGFP result in a cleavage product, demonstrating the protein’s activity. Levels of the cleavage product are similar to the levels generated using our Cas9 All-in-one System, and no activity is seen in the absence of Cas9. Figure 6B shows a similar set of studies for NLS-Cas9-EGFP electroporated into target cells.

Figure 6. NLS-Cas9-EGFP protein is functional in an in vivo assay.

FAQs

Resources

Brochure: CRISPR/Cas9 Products and Services

Protocol: Cas9 Protein Transfection with gRNA

Citations