PurposeExpression plasmid for human codon-optimized increased fidelity HeFm1SpCas9 (without U6-sgRNA coding sequence)
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||92110||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
This material is available to academics and nonprofits only.
Vector backbonepX330-like (without U6-sgRNA coding sequence)
- Total vector size (bp) 8077
Vector typeMammalian Expression, CRISPR
Growth in Bacteria
Growth Strain(s)NEB Stable
Copy numberHigh Copy
Gene/Insert name3xFLAG-NLS-Streptococcus pyogenes Highly enhanced Fidelity mut1 Cas9-NLS
Insert Size (bp)4272
MutationR661A, Q695A, K848A, Q926A, K1003A, R1060A
- Promoter Cbh
/ Fusion Proteins
- 3xFLAG (N terminal on insert)
- NLS (N terminal on insert)
- NLS (C terminal on insert)
- Cloning method Unknown
- 5′ sequencing primer AAGGGATGGTTGGTTGGTGG
- 3′ sequencing primer GGAAAGGACAGTGGGAGTGG (Common Sequencing Primers)
Terms and Licenses
pCbh-3xFLAG-NLS-HeFm1SpCas9-NLS (without U6-sgRNA coding sequence).
Highly enhanced Fidelity nuclease variant HeFm1SpCas9 contains mutations from both eSpCas9 (1.1) and SpCas9-HF1.
HeFSpCas9s exhibits activity with spectacularly increased specificity specifically for those targets that are met with higher off-target propensity by eSpCas9 (1.1) and SpCas9-HF1.
The lack of sgRNA expression cassette allows for easy testing of various SpCas9 variants with the same sgRNA expressed from a separate plasmid (e.g. pmCherry_gRNA, Addgene# #80457).
For detailed information and plasmid usage, please see the associated publication.
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:HeFm1SpCas9 was a gift from Ervin Welker (Addgene plasmid # 92110 ; http://n2t.net/addgene:92110 ; RRID:Addgene_92110)
For your References section:Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage. Kulcsar PI, Talas A, Huszar K, Ligeti Z, Toth E, Weinhardt N, Fodor E, Welker E. Genome Biol. 2017 Oct 6;18(1):190. doi: 10.1186/s13059-017-1318-8. 10.1186/s13059-017-1318-8 [pii] PubMed 28985763