PurposeThe 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
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||65779||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
Backbone manufacturerJoung lab (Addgene Plasmid # 43860)
Vector typeMammalian Expression, CRISPR
Growth in Bacteria
Growth Strain(s)XL1 Blue
Copy numberHigh Copy
Gene/Insert nameSaCas9 gRNA backbone, without spacer sequence
- Promoter U6
- Cloning method Gibson Cloning
- 5′ sequencing primer OS280 (5'-CAGGGTTATTGTCTCATGAGCGG-3') (Common Sequencing Primers)
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).
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:VVT1 was a gift from Keith Joung (Addgene plasmid # 65779 ; http://n2t.net/addgene:65779 ; RRID:Addgene_65779)
For your References section:Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK. Nature. 2015 Jun 22. doi: 10.1038/nature14592. 10.1038/nature14592 PubMed 26098369