LUHMES Cell Editing
LUHMES cell gene editing will must be carried out on the parental cell line provided by the Genomic Toxicology Lab. To knock-in HiBiT tag vendor must design CRISPR guide RNAs with low off target potential. Based on designed gRNA vendor will also design the donor template for tag knock-in at specified sites. Prior to gene editing cells will be qualified, CRISPR gene editing will be via electroporation. Following electroporation, cells will be allowed to recover and
undergo pool analysis for gene knock-in. Then single cell clones will be expanded, and reanalyzed for knock-in at the specified gene locus. Two independent colonies of successfully edited cells will be frozen in two vials each and the vials shipped to the NCATS Genomic Toxicology Lab.
The vendor will perform this CRISPR gene editing in steps:
• Qualify cells prior to gene editing
• Design CRISPR quide RNA
• Design donor template for specified genes with HiBiT tag
• Optimize and Perform electroporation
• Perform pool analysis
• Perform single cell clonal expansion
• Perform clonal analysis
• Qualify cells after gene editing for knock-in at specified sites, pluripotency and karyotype
• Provide cryopreserved reporter cells and QC data to NCATS.

Specified gene target;
MT1-G transcript, Ensembl accession: ENST00000444837.6