gRNA-CasRx_SD1-pSV40-TagBFP
(Plasmid #221004)

• Purpose: Transiently express CasRx DR30 repeat with gRNA spacer. Contains SD1 mutation to enhance efficiency. SV40-TagBFP cassette to monitor transfection efficiency. Use BbsI with overhangs Fw-AAAC, Rv-AAAA.
• Depositing Labs: Benjamin Blencowe, Mikko Taipale
• Publication: Efficient, specific, and combinatorial control of endogenous exon splicing with dCasRx-RBM25 (unpublished)

Backbone

• Vector backbone: pXR003 (Addgene #109053)
• Vector type: Mammalian Expression, CRISPR
• Selectable markers: tagBFP

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: CasRx 30nt processed direct repeat with SD1 mutation
• gRNA/shRNA sequence: gRNA spacer
• Species: H. sapiens (human)
• Mutation: DR1 A>T
• Promoter: U6

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: unknown (unknown if destroyed)

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Evrogen Limited Use Label License for FPs
• Industry Terms:
  • Not Available to Industry

Depositor Comments

The gRNA backbone contains the SD1 mutation (DR1 A>T) in the direct repeat, which boosted knockdown efficiency in a previous paper (Wessels et al., 2020, Nat Biotech). We have found increased splicing modulation with this mutation compared to the WT direct repeat.