AICSDP-45: TTN-mEGFP
(Plasmid #114412)

• Purpose: Homology arms and linker-mEGFP sequence for C-terminus tagging of human TTN, via Cas9-excisable CAGGS-mCherry selection cassette
• Depositing Lab: Allen Institute for Cell Science
• Publication: Roberts et al Stem Cell Reports. 2019 May 14;12(5):1145-1158. doi: 10.1016/j.stemcr.2019.03.001. Epub 2019 Apr 4.

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: TTN Homology Arms with linker-mEGFP and Cas9-excisable selection cassette
• Species: H. sapiens (human)
• Insert Size (bp): 5709
• Mutation: homology arms contain point mutations to disrupt crRNA binding sites used, and WTC-specific homozygous SNP(s)
• Entrez Gene: TTN (a.k.a. CMD1G, CMH9, CMPD4, CMYO5, CMYP5, EOMFC, HMERF, LGMD2J, LGMDR10, MYLK5, SALMY, TMD)
• Tag / Fusion Protein:
  • linker-mEGFP (C terminal on insert)

Cloning Information

• Cloning method: Unknown

Resource Information

• Supplemental Documents:
  • annotated partial sequence from depositor

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 TTN 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 fluorsecence-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 the bioRxiv preprint.