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Two Vector CRISPR/Cas9 System

The Two Vector CRISPR system can be used to perform functional CRISPR, CRISPRi, or CRISPRa screens in vivo or in vitro by expressing sgRNA (single guide RNA, or gRNA) and Cas9 or dCas9 from different lentiviral vectors.

Stable Expression of Cas9/dCas9 and sgRNA

  • Pre-transduce with Cas9 or dCas9 lentivirus to decrease noise in your knockout screen
  • Make high-titer sgRNA lentivirus for efficient transduction
  • Vectors contain different antibiotic resistance genes to ensure stability and easy selection
  • Tet-inducible and constitutive U6 expression available

Lentiviral sgRNA Libraries Available

Cas9 or dCas9 Expression Vectors

  • Cas9 nuclease for knockout, dCas9-KRAB for CRISPRi, or dCas9-VPH for CRISPRa expressed from a separate vector
  • Choose from several antibiotic selection markers, including blasticidin and hygromycin resistance
CRISPR Two Vector CRISPR Cas9 sgRNA gRNA Tet-inducible knockout
Two Vector CRISPR Cas9 U6 knockout

CRISPR two-vector constitutive or tet-inducible knockout

The Two Vector CRISPR system is available with both (a) tet-inducible or (b) constitutive expression of the sgRNA. GFP knockout constructs were built using either the tet-inducible or constitutive sgRNA expression vector and transduced in cells stably expressing both GFP and Cas9. In cells containing tet-inducible constructs, upon addition of doxycycline, over 80% of the GFP is knocked out. Knockout using constitutive expression of GFP sgRNA is effective by 9 days post-transduction and increases in efficiency after 12 days.

CRISPR Two Vector CRISPR Cas9 sgRNA gRNA Tet-inducible knockout        Two Vector CRISPR Cas9 U6 knockout

The Two Vector CRISPR system is available with both (a) tet-inducible or (b) constitutive expression of the sgRNA. GFP knockout constructs were built using either the tet-inducible or constitutive sgRNA expression vector and transduced in cells stably expressing both GFP and Cas9. In cells containing tet-inducible constructs, upon addition of doxycycline, over 80% of the GFP is knocked out. Knockout using constitutive expression of GFP sgRNA is effective by 9 days post-transduction and increases in efficiency after 12 days.

Cellecta Two Vector CRISPR system includes separate sgRNA and Cas9 vectors

Vector maps for the Two Vector CRISPR/Cas9 system, consisting of separate sgRNA expression vector and Cas9 expression vector.

Cellecta pRSIT16-U6Tet-sh-HTS6-CMV-TetRep-2A-TagRFP-2A-Puro Map

Vector map for the Tet-inducible sgRNA vector (8.7 kb)

User Manuals

  • Packaging, Titering, and Transduction of Lentiviral Constructs, v9a (PDF)
  • Cloning of shRNA and sgRNA Templates into Cellecta Expression Vectors (PDF)

How to Order

To place an order or simply calculate pricing, please fill out the Two-Vector CRISPR Construct Order Form and email it to us at orders@cellecta.com. Please indicate whether you are placing an order or requesting a quote. If placing an order, please refer to the PO number and email the PO to orders@cellecta.com or fax it to 650-938-3911.

For our selection of positive and negative CRISPR control constructs, please see our Control Constructs web page.

Required Products:

Catalog #Item DescriptionPrice
SVC9-PSpR-CMV-Cas9-2A-Hygro Cas9 Expression Vector (plasmid), 25 μg$300
SVC9-VSpR-CMV-Cas9-2A-Hygro Cas9 Expression Vector (virus), 1 x 106 TU$500
SVC9B-PSpR-CMV-Cas9-2A-Blast Cas9 Expression Vector (plasmid), 25 μg$300
SVC9B-VSpR-CMV-Cas9-2A-Blast Cas9 Expression Vector (virus), 1 x 106 TU$500
SVC9G-PSpR-CMV-Cas9-2A-GFP Cas9 Expression Vector (plasmid), 25 μg$300
SVC9G-VSpR-CMV-Cas9-2A-GFP Cas9 Expression Vector (virus), 1 x 106 TU$500

Related Products:

Catalog #Item DescriptionPrice
CPCP-K2AReady-to-Use Lentiviral Packaging Plasmid Mix, 250 μg
(for (25) 10-cm plates)
$325
LFVC1LentiFuge™ Viral Concentration Reagent, 1 ml
(for 1 liter supernatant)
$325
CRTESTCRISPRtest™ Functional Cas9 Activity Kit (for 5 cell line pairs)$750