HeFSpCas9
(Plasmid
#92355)
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PurposeExpression plasmid for human codon-optimized increased fidelity HeFSpCas9 (without U6-sgRNA coding sequence)
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 92355 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backbonepX330-like (without U6-sgRNA coding sequence)
- Total vector size (bp) 8077
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Vector typeMammalian Expression, CRISPR
Growth in Bacteria
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Bacterial Resistance(s)Ampicillin, 100 μg/mL
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Growth Temperature37°C
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Growth Strain(s)NEB Stable
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Copy numberHigh Copy
Gene/Insert
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Gene/Insert name3xFLAG-NLS-Streptococcus pyogenes Highly enhanced Fidelity Cas9-NLS
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Alt namepX330-Flag-HeFSpCas9
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SpeciesS. pyogenes
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Insert Size (bp)4272
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MutationN497A, R661A, Q695A, K848A, Q926A, K1003A, R1060A
- Promoter Cbh
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Tags
/ Fusion Proteins
- 3xFLAG (N terminal on insert)
- NLS (N terminal on insert)
- NLS (C terminal on insert)
Cloning Information
- Cloning method Unknown
- 5′ sequencing primer TAGCTGAGCAAGAGGTAAGG (Common Sequencing Primers)
Terms and Licenses
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Academic/Nonprofit Terms
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Industry Terms
- Not Available to Industry
Trademarks:
- Zeocin® is an InvivoGen trademark.
Depositor Comments
pCbh-3xFLAG-NLS-HeFSpCas9-NLS (without U6-sgRNA coding sequence).
Highly enhanced Fidelity nuclease variant HeFSpCas9 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.
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For your Materials & Methods section:
HeFSpCas9 was a gift from Ervin Welker (Addgene plasmid # 92355 ; http://n2t.net/addgene:92355 ; RRID:Addgene_92355) -
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