pRM053 His-TEV-Cas9 triNLS 0C
(Plasmid #196244)

• Purpose: Expression of NLS-rich Cas9 (0 Cys residues, mammalian codon optimized)
• Depositing Lab: Ross Wilson
• Publication: Foss et al Nat Biomed Eng. 2023 May;7(5):647-660. doi: 10.1038/s41551-023-01032-2. Epub 2023 Apr 25.

Backbone

• Vector backbone: pEC-K-MBP
• Backbone manufacturer: pET-based custom vector
• Backbone size w/o insert (bp): 6400
• Total vector size (bp): 9073
• Modifications to backbone: MBP removed
• Vector type: Bacterial Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: Cas9
• Alt name: Cas9-0C-triNLS
• Species: Streptococcus pyogenes
• Insert Size (bp): 4102
• Mutation: Cys at positions 80 and 574 have been mutated to Ser
• Tags / Fusion Proteins:
  • His6 (N terminal on backbone) (N terminal on insert)
  • HA (C terminal on insert)
  • SV40 (C terminal on insert)
  • Nucleoplasmin (C terminal on insert)
  • NLS (N terminal on insert)

Cloning Information

• Cloning method: Ligation Independent Cloning
• 5′ sequencing primer: T7 promoter
• 3′ sequencing primer: T7 terminator

Resource Information

• Supplemental Documents:
  • pRM053_02.gbk
• A portion of this plasmid was derived from a plasmid made by: pMJ806 was a gift from Jennifer Doudna (Addgene plasmid # 39312 ; http://n2t.net/addgene:39312 ; RRID:Addgene_39312)

Terms and Licenses

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

Depositor Comments

This submission's cysteine-free triNLS Cas9 construct bears an unexpected and unintentional point mutation in the Cas9 sequence, H1311N, that was not identified until after deposition. Analysis of the Cas9 structure suggests that this mutation is unlikely to impact enzyme function or properties. H1311 is located in the PAM-interacting domain (PID), a relatively rigid part of the Cas9 structure. In the Cas9 crystal structure (PDB ID 4UN3), the side chain of H1311 is solvent exposed and does not interact with other Cas9 amino acids or bound nucleic acid. Substitution of histidine with asparagine (a hydrophilic amino acid) should be well tolerated. We do not anticipate any consequences associated with this mutation. This mutation is reflected in the sequence files currently associated with this Addgene entry. Note that our Addgene submission 403489 (triNLS Cas9 with two cysteines) is very similar to this plasmid but it does not contain the H1311N mutation (it has H1311).