AICSDP-55:DMD-mEGFP
(Plasmid #159744)

• Purpose: Homology arms and linker-mEGFP sequence for C-terminus tagging of human DMD via Cas9-excisable CAGGS-mCherry selection cassette
• Depositing Lab: Allen Institute for Cell Science
• Publication: Allen Institute for Cell Sciences Plasmids (unpublished)

Backbone

• Vector backbone: pUC57
• Vector type: Mammalian Expression, CRISPR ; Donor Template

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: DMD Homology Arms with linker-mEGFP and Cas9-excisable selection cassette
• Alt name: DMD
• Species: H. sapiens (human)
• Insert Size (bp): 6410
• Mutation: homology arms contain point mutations to disrupt crRNA binding sites used, and WTC-11 specific homozygous SNP(s)
• Entrez Gene: DMD (a.k.a. BMD, CMD3B, DXS142, DXS164, DXS206, DXS230, DXS239, DXS268, DXS269, DXS270, DXS272, MRX85)
• Tag / Fusion Protein:
  • linker-mEGFP (C terminal on insert)

Cloning Information

• Cloning method: Unknown

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

This plasmid has been used with locus-specific CRISPR/Cas9 in a two-step editing process to add a mEGFP tag to the C-terminus of human DMD in WTC human induced pluripotent stem cells by the Allen Institute for Cell Science. After initial HDR, stable expression of CAGGS-driven mCherry will indicate cells with integrated donor sequence. This selection cassette is designed to be removed with subsequent CRISPR/Cas9-mediated excision as previously described (https://doi.org/10.1101/342881) A linker of 3-21 amino acids will be produced after excision. To enrich for edited cells, see our protocol for fluorescence-assisted cell sorting and subcloning of hiPSCs (https://www.allencell.org/instructional-videos-and-tutorials-for-cell-methods.html) Further, we recommend PCR-based assays for identifying precisely edited clones as previously described (https://www.molbiolcell.org/doi/abs/10.1091/mbc.e17-03-0209) For more information on the entire plasmid collection, please see https://www.addgene.org/allen-institute-cell-science/ . Please visit https://www.biorxiv.org/content/early/2018/06/09/342881 for BioRxiv preprint