CMV-hspCas9(D10A)-T2A-Puro Two Vector Cas9 SmartNickaseTM Lentivector

When working with difficult-to-transfect-cells and you want to reduce off-target events, use our lentiviral Cas9 Nickase—CMV-hspCas9(D10A)-T2A-Puro.

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Cas9 expression vector for the Two Vector SmartNickase System (CMV-hspCas9(D10A)-T2A-Puro Lentivector Plasmid)

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Cas9 expression vector for the Two Vector SmartNickase System (CMV-hspCas9(D10A)-T2A-Puro Lentivector Plasmid)

[variation_id] => 63400 [variation_is_active] => 1 [variation_is_visible] => 1 [weight] => [weight_html] => N/A )
10 µg
CASLV200PA-1
$ 595

KIT: Cas9 expression vector for the Two Vector SmartNickase System (CMV-hspCas9(D10A)-T2A-Puro Lentivector Plasmid) plus LentiStarter Packaging Kit

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KIT: Cas9 expression vector for the Two Vector SmartNickase System (CMV-hspCas9(D10A)-T2A-Puro Lentivector Plasmid) plus LentiStarter Packaging Kit

[variation_id] => 63401 [variation_is_active] => 1 [variation_is_visible] => 1 [weight] => [weight_html] => N/A )
1 Kit
CASLV200PA-KIT
$ 995

Cas9 expression vector for the Two Vector SmartNickase System (CMV-hspCas9(D10A)-T2A-Puro LentivectorPre-Packaged Lentiviral Particles (>10^6 IFUs))

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Cas9 expression vector for the Two Vector SmartNickase System (CMV-hspCas9(D10A)-T2A-Puro LentivectorPre-Packaged Lentiviral Particles (>10^6 IFUs))

[variation_id] => 63402 [variation_is_active] => 1 [variation_is_visible] => 1 [weight] => [weight_html] => N/A )
2 x 25 µL
CASLV200VA-1
$ 595
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Overview

On-target genome editing in transfection-resistant cells

When you’re genome editing in a transfection-resistant cell line and need to keep off-target events to a minimum, turn to one of SBI’s Cas9 SmartNickaseTM Lentivector Systems. Unlike the wildtype Cas9 protein which introduces double-strand breaks (DSBs), the CMV-hspCas9(D10A)-T2A-Puro Two Vector 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.

CMV-hspCas9(D10A)-T2A-Puro Two Vector Cas9 SmartNickase Lentivector

Available as lentivector plasmids, ready-to-transduce pre-packaged pseudovirus, and in a package-your-own-lentivector kit with the lentivector plasmid and the LentiStarter 3.0 Packaging Kit, the CMV-hspCas9(D10A)-T2A-Puro Two Vector Cas9 SmartNickase Lentivector expresses human codon-optimized Cas9 nickase (the D10A mutant) from the strong CMV promoter and includes the puromycin selection marker.

  • Conduct genome editing and engineering in difficult-to-transfect cell lines
  • Stay more on-target with Cas9 nickase activity
  • Drive Cas9 expression from the CMV promoter, for high expression in many commonly-used cell lines (HeLa, HEK293, HT1080)
  • Perform in vivo engineering of model organisms
  • Supports synthetic biology applications, gene- and cell-based therapy development, and genome-wide functional screening

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 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?

Use this table to choose the CRISPR/Cas9 product that’s right for you:

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.