B-HypaSpCas9
(Plasmid #126763)

• Purpose: Expression plasmid for human codon-opt. increased fidelity Blackjack-HypaSpCas9 (w/o U6-sgRNA). Cleaves both 20nt and 5'-extended 21nt spacer containing sgRNAs with higher fidelity than HypaSpCas9.
• Depositing Lab: Ervin Welker
• Publication: Kulcsar et al Nat Commun. 2020 Mar 6;11(1):1223. doi: 10.1038/s41467-020-15021-5.

Backbone

• Vector backbone: pX330-like (without U6-sgRNA coding sequence)
• Total vector size (bp): 8056
• Vector type: Mammalian Expression, CRISPR

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: B-HypaSpCas9
• Alt name: Blackjack-HypaSpCas9
• Species: S. pyogenes
• Insert Size (bp): 4251
• Mutation: N692A; M694A; Q695A; H698A; amino acids 1005-1013 replaced with two glycine
• Promoter: CBh
• Tags / Fusion Proteins:
  • 3xFLAG (N terminal on insert)
  • NLS (N terminal on insert)
  • NLS (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: MluI (not destroyed)
• 3′ cloning site: Pfl23II (destroyed during cloning)
• 5′ sequencing primer: GAACATCGTGATCGAAATG
• 3′ sequencing primer: TGTCGAAGCCGCCGTAC

Terms and Licenses

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

Depositor Comments

pCbh-3xFLAG-NLS-Blackjack-HypaSpCas9-NLS (without U6-sgRNA coding sequence, with silent mutations).

Blackjack variants cleave both 20nt and 5'-extended 21nt spacer containing sgRNAs. B-HypaSpCas9 shows higher fidelity and target selectivity than its parental increased fidelity variant, HypaSpCas9.

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.