EF1α-hspCas9-nickase-H1-gRNA All-in-one Cas9 SmartNickase Plasmid (circular)

Use this first-generation SBI EF1α-hspCas9 SmartNickase when you want to reduce off-target events, have transfectable cells, and prefer an intact vector.

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All-in-one Cas9 Nickase: EF1α-hspCas9-nickase-H1-gRNA SmartNickase vector

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All-in-one Cas9 Nickase: EF1α-hspCas9-nickase-H1-gRNA SmartNickase vector

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10 µg
CAS800A-1
$ 487
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Overview

Stay on-target with your genome editing projects

For pre-clinical and other applications where you need to minimize off-target Cas9 activity and are using transfectable cells, All-in-one Cas9 SmartNickase plasmids are an excellent choice. Unlike our newer Cas9 constructs, this first generation Cas9 SmartNickase—EF1α-hspCas9-nickase-H1-gRNA All-in-one Cas9 SmartNickase Plasmid—does not contain a selectable marker for identifying transfected cells and is now available as an intact, circular plasmid.

EF1α-hspCas9-nickase-H1-gRNA All-in-one Cas9 SmartNickase Plasmid (circular)

Unlike the wildtype Cas9 protein which introduces double-strand breaks (DSBs), the Cas9 SmartNickase introduces paired nicks at the gRNA-directed site. Creating nicks favors the higher-fidelity homologous recombination process over non-homologous end joining (NHEJ), with paired nicking shown to reduce off-target activity by 50- to 1,500-fold in cell lines, and to facilitate gene knockout in mice without losing on-target cleavage efficiency1.

  • Conveniently deliver Cas9 Nickase and gRNA with a single vector
  • Reduce off-target activity
  • Drive Cas9 Nickase expression with the EF1α promoter, which provides medium expression levels in most cell types, including primary cells and stem cells
  • Express gRNA from the H1 promoter for maximum specificity and choice of targets
  • Ensure efficient import of Cas9 Nickase to the nucleus with N-term and C-term nuclear localization signals (NLSs)
  • Boost Cas9 Nickase gene expression and stabilize the transcript via the WPRE regulatory element after the C-term NLS
  • Easily detect and/or purify the Cas9 Nickase protein with the N-term myc-tag

As with all of our Cas9 Nickase delivery options, the EF1α-hspCas9-nickase-H1-gRNA Plasmid is functionally validated and comes backed by our expert technical support team—if you’ve got a genome engineering question just ask by emailing tech@systembio.com.

Why an HR targeting vector is a recommended

Even though gene knock-outs can result from DSBs caused by Cas9 alone, SBI recommends the use of HR targeting vectors (also called HR donor vectors) for more efficient and precise mutation. HR donors can supply elements for positive or negative selection ensuring easier identification of successful mutation events. In addition, HR donors can include up to 6-8 kb of

Even though gene knock-outs can result from DSBs caused by Cas9 alone, SBI recommends the use of HR targeting vectors (also called HR donor vectors) for more efficient and precise mutation. HR donors can supply elements for positive or negative selection ensuring easier identification of successful mutation events. In addition, HR donors can include up to 6-8 kb of open reading frame for gene knock-ins or tagging, and, when small mutations are included in either 5’ or 3’ homology arms, can make specific, targeted gene edits.

Not sure whether you need a CRISPR/Cas9 plasmid, purified protein, or mRNA?

For This Application Use These Products
MODIFYING ORGANISMS Use These Products Use These Products
·       Gene tagging

·       Transgenic organism generation

·       Model organism engineering

Creating transgenic animals ·       Injectable Cas9 mRNA &

·       gRNA Synthesis Kits

·       Purified Cas9 Protein

In vivo genome editing in animal models ·       AAV-Cas9 Vectors

·       Purified 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

·       Purified Cas9 Protein

·       AAV-Cas9 VectDifficult to transfect cell lines,

·       Primary cells

·       Hematopoietic cells

·       Stem cellsors

·       Lenti-Cas9 System

·       AAV-Cas9 Vectors

·       Lenti-Cas9 System

SCREENING
·       Genome-wide surveys

·       gRNA library screens

·       Functional screens

All applications requiring

stable Cas9 overexpression

·       Lenti-Cas9 System

·       AAVS1 Safe Harbor Cas9

·       Knock-In System

·       Purified Cas9 Protein

PRE-CLINICAL APPLICATIONS
·       Off-target events are of highest concern All applications ·       Cas9 Nickase, available in all delivery formats

·       Purified Cas9 Protein

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

References

  1. Ran, FA. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013 Oct 24; 8:2281-2308. PMCID: PMC3969860.

How It Works

Genome engineering with CRISPR/Cas9

For 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.

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

Validating SBI’s CRISPR/Cas9 SmartNickase and Null Nuclease Vectors

The All-in-one Cas9 SmartNickase Vector shows genome editing activity

Figure 1. The All-in-one Cas9 Null Nuclease Vector shows no genome editing activity. We compared the activity of wild-type Cas9 (EF1α-hspCas9-H1-gRNA All-in-one SmartNuclease Plasmid, Cat.# CAS900A-1), Cas9 SmartNickase (EF1α-hspCas9-nickase-H1-gRNA All-in-one SmartNickase Plasmid, Cat.# CAS800A-1), and Cas9 Null Nuclease (EF1α-hspCas9-DM-H1-gRNA All-in-one Cas9 Null Nuclease Plasmid, Cat.# CAS805A-1) to insert a GFP reporter at the AAVS1 site through the use of AAVS1-targeting gRNA and an AAVS1-targeting HR Targeting Vector (AAVS1 Safe Harbor cDNA/miRNA Targeting HR Donor Vector (pAAVS1D-PGK-MCS-EF1α-copGFPpuro), Cat.# GE602A-1).
(Left panel) Fluorescence (left-most column) and bright field (right-most column) microscopy showing that both Cas9 SmartNuclease (top set) and Cas9 SmartNickase (middle set) are able to insert GFP into the genome, whereas the Cas9 Null Nuclease is not (bottom set).
(Right panel) A Surveyor Nuclease Assay corroborates the lack of genome editing capabilities of the Cas9 Null Nuclease. The lack of cleavage seen in the SmartNickase lane is likely due to the lower frequency of GFP insertion.


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