VVT1
(Plasmid #65779)

• Purpose: The Joung Lab recommends using BPK2660 (Addgene plasmid 70709) instead of VVT1 as guide RNAs cloned into BPK2660 are more effective than this plasmid. Human expression plasmid for S. aureus Cas9 sgRNA
• Depositing Lab: Keith Joung
• Publication: Kleinstiver et al Nature. 2015 Jun 22. doi: 10.1038/nature14592.

Backbone

• Vector backbone: MLM3636
• Backbone manufacturer: Joung lab (Addgene Plasmid # 43860)
• Vector type: Mammalian Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): XL1 Blue
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: SaCas9 gRNA backbone, without spacer sequence
• Species: Synthetic
• Promoter: U6

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: OS280 (5'-CAGGGTTATTGTCTCATGAGCGG-3')

Resource Information

• Supplemental Documents:
  • Annotated Genbank file
  • Joung Lab gRNA Cloning Protocol
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

Use BsmBI sites to insert sgRNA spacer sequence into the vector. See the supplemental document "Joung Lab gRNA Cloning Protocol" for more details.

UPDATE 5/31/2016: The Joung Lab recommends using BPK2660 (Addgene plasmid # 70709) instead of VVT1 as an SaCas9 guide RNA cloning vector, as guide RNAs cloned into BPK2660 are more effective than those cloned into VVT1. The difference in efficacy is due to VVT1 containing the full length guide RNA (predicted from the natural CRISPR sequence in S. aureus), whereas BPK2660 uses a truncated gRNA that is more effective. For the same oligo/spacer, the rate of mutagenesis is greater using BPK2660. See Kleinstiver et al., 2015 Nat Biotech for additional information on the truncated gRNA plasmid (https://www.ncbi.nlm.nih.gov/pubmed/26524662)