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MSCV-hspCas9-T2A-Puro-H1-gRNA All-in-one Cas9 SmartNuclease™ Lentivector

Get the convenience of all-in-one Cas9 & gRNA delivery in a lentivector and streamline genome editing in hard-to-transfect-cells—MSCV-hspCas9-T2A-Puro-H1-gRNA.

  • Conduct genome editing and engineering in difficult-to-transfect cell lines
  • Simultaneously deliver Cas9 and gRNA from a single lentivector
  • Drive Cas9 expression from the MSCV promoter, for high expression in hematopoietic and stem cells
  • Perform in vivo engineering of model organisms
  • Supports synthetic biology applications, gene- and cell-based therapy development, and genome-wide functional screening
Catalog Number
Description
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Price
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CASLV320PA-1
All-in-one MSCV-hspCas9-T2A-Puro-H1-gRNA linearized all-in-one SmartNuclease Lentivector Plasmid
10 Reactions
$ 695

Overview

Genome editing in transfection-resistant cells

When you want to create stable Cas9 editing cell lines and/or would like to edit the genome of a cell line that is resistant to transfection by plasmids, turn to one of SBI’s Cas9 SmartNuclease Lentivector Systems. Available as pre-linearized, ready-to-clone lentivector plasmids, the MSCV-hspCas9-T2A-Puro-H1-gRNA All-in-one Cas9 SmartNuclease™ Lentivector expresses human codon-optimized Cas9 wild-type nuclease from the strong MSCV promoter, includes the puromycin selection marker, and delivers gRNA from an H1 promoter.

  • Conduct genome editing and engineering in difficult-to-transfect cell lines
  • Simultaneously deliver Cas9 and gRNA from a single lentivector
  • Drive Cas9 expression from the MSCV promoter, for high expression in hematopoietic and stem cells
  • 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
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

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

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.

Have Questions?

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