pXPR_011
(Plasmid #59702)

• Purpose: This lentiviral vector can be used to assay Cas9 activity.
• Depositing Labs: John Doench, David Root
• Publication: Doench et al Nat Biotechnol. 2014 Sep 3. doi: 10.1038/nbt.3026.

Backbone

• Vector backbone: pLKO.1
• Total vector size (bp): 8395
• Vector type: Mammalian Expression, Lentiviral, CRISPR
• Selectable markers: Puromycin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Stbl3
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: EGFP sgRNA
• Species: Synthetic
• Promoter: hU6

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: LKO.1 5'

Resource Information

• Articles Citing this Plasmid:
  • 30 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

This lentiviral vector can be used to assay Cas9 activity. In the absence of Cas9, the cells are both resistant to puromycin and express EGFP. In the presence of functional levels of Cas9, the sgRNA expressed from the U6 cassette targets EGFP, and thus cells will still be puromycin-resistant, but will no longer express EGFP. The fraction of EGFP-positive and -negative cells can be accurately quantitated by flow cytometry. Please note that it is unlikely that 100% of cells will ever be EGFP-negative, as a fraction of cells are likely to have in-frame indels, and those the cells may still be green. This assay is best done with cells infected with pXPR_011 at an MOI <1.