pAAV-MP-84-spCas9-U6-gRNA-tlock
(Plasmid #206936)

• Purpose: Expression of spCas9 endonuclease and gRNA in an AAV vector allowing packaging of viral genome smaller than 5 kb.
• Depositing Lab: Markus Grompe
• Publication: Grompe Lab Cas9 Plasmids 2023 (unpublished)

Backbone

• Vector backbone: pAAV-MP-84
• Backbone manufacturer: Sunghee Chai, Markus Grompe
• Backbone size w/o insert (bp): 2963
• Vector type: Mammalian Expression, AAV

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): NEB Stable
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: spCas9
• Species: S. Pyogenes
• Insert Size (bp): 4101
• Promoter: MP-84
• Tags / Fusion Proteins:
  • SV40 NLS (N terminal on insert)
  • SV40 NLS (C terminal on insert)

Cloning Information

• Cloning method: Ligation Independent Cloning

Resource Information

• Supplemental Documents:
  • pAAV-MP-84-spCas9-U6-gRNA-tlock
• A portion of this plasmid was derived from a plasmid made by: Addgene plasmid #60957, Feng Zhang Lab

Terms and Licenses

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

Depositor Comments

The DNA sequence of spCas9 including SV40 NLS tag (no HA tag) is from the pX551 plasmid sequence (Feng Zhang lab). spCas9 is expressed from MP-84 promoter and the gRNA sequence from a shorter version of human U6 promoter (miniU6). The gRNA contains a locked tracrRNA (t-lock) secondary structure (Riesenberg et al., Nat. Commun., 2022, 13:489).