lentiGuide-Crimson
(Plasmid #70683)

• Purpose: Expresses S. pyogenes CRISPR chimeric RNA element with customizable sgRNA from U6 promoter and Crimson reporter from EF-1a promoter. Lentiviral backbone.
• Depositing Lab: Daniel Bauer
• Publication: Canver et al Nature. 2015 Sep 16. doi: 10.1038/nature15521.

Backbone

• Vector backbone: Custom
• Backbone size w/o insert (bp): 9482
• Total vector size (bp): 10260
• Vector type: Mammalian Expression, Lentiviral, CRISPR
• Selectable markers: Crimson Reporter

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): NEB Stable
• Growth instructions: Only amplify in RecA- bacteria (eg. Stbl3).
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: S. pyogenes sgRNA cassette
• Alt name: S. pyogenes CRISPR customizable RNA element
• Species: Synthetic
• Insert Size (bp): 100
• Promoter: hU6

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: BsmBI (not destroyed)
• 3′ cloning site: N/A (unknown if destroyed)
• 5′ sequencing primer: U6 [5'-GAGGGCCTATTTCCCATGATT-3']

Gene/Insert 2

• Gene/Insert name: Crimson Reporter
• Species: Synthetic
• Insert Size (bp): 678
• Promoter: Ef1-a

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: BsiWI (not destroyed)
• 3′ cloning site: MluI (not destroyed)
• 5′ sequencing primer: EF1a-F [5'-TCAAGCCTCAGACAGTGGTTC-3']

Resource Information

• Supplemental Documents:
  • pHKO_09 - pLKO-U6-BsmBI-chRNA(+85)-EF1a-Crimson-FILLER
• Addgene Notes:
  • Addgene Diagnostic Digest
• Articles Citing this Plasmid:
  • 5 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Cloned in Crimson Reporter in place of PURO reporter into lentiGuide-Puro.
Improved vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047. 10.1038/nmeth.3047 PubMed 25075903

Canver MC, Smith EC, Sher F, Pinello L, Sanjana NE, Shalem O, Chen DD, Schupp PG, Vinjamur DS, Garcia SP, Luc S, Kurita R, Nakamura Y, Fujiwara Y, Maeda T, Yuan G, Zhang F, Orkin SH, Bauer DE. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature. 2015 September 16; in press; doi:10.1038/nature15521.