UA Cancer Center Genome Editing Facility

Overview of Services

The Genome Editing Facility (GEF) provides novel cell line models based on CRISPR/Cas9 technology. The services offered by the GEF include:

• knock-in mutation
• knock-out mutation
• gene product tagging at endogenous locus.
• safe-harbor gene insertions

The service allows the investigator to manipulate the genome of nearly any cell line that can be transfected and cloned. The GEF uses a “hit and run” approach, in which cells are transfected with Cas9 protein and CRISPR guide RNA. Transfected cells are then cloned by flow sorting, and the clones are screened for the desired mutation by PCR-based methods. One major advantage of the hit and run approach over vector-based approaches is that after editing there are no additional sequences, such as E. coli shuttle vector sequences or exogenous eukaryotic promoters left in the editing cells. These additional sequences could change the normal regulation of gene expression of your gene of interest.

The most widely used GEF services include (i) single exon to multiple exon deletions to create loss-of-function mutations in a gene of interest and (ii) single-to multiple-nucleotide DNA substitutions or insertion/deletions to create amino acid substitutions, such as phospho-mutants. Loss-of-function mutations allow the investigator to study the genetic function of a gene product. Amino acid substitution mutations can be “separation-of-function mutations” that allow the investigator to study functional effects of expressed proteins that have altered activities. One could also introduce promoter and enhancer mutations in which the mutation changes gene function in cis.

The GEF offers protein tagging of your gene of interest. The tag could be fluorescent-protein tag, such as GFP, or another sort of peptide tag, such as a myc, HA tag, or a HiBiT (Promega) tag for antibody free protein tagging. The tag could be place at the N- or C-terminus of the protein. This service allows the investigator to study the localization of the protein where the tagged form of the gene is expressed from its endogenous genomic site and from its natural promoter. Tagging can be useful in drug development.

The GEF also creates cell lines that contain your gene of interest, with a tag, inserted at the safe harbor locus AAVS1 and with its transcriptional expressed controlled by inducer such as doxycycline. Cells modified in this way can be useful for the study of the effects of expression of the protein at higher levels and for the study of essential genes, where the cells cannot proliferate in the absence of a gene product.

In most cases, the GEF provides multiple homozygous mutant clones, and also available are various heterozygous and genetic compound clones at the request of the investigator.

Leadership

Nathan Ellis            GEF Director          520-626-7979

Location and hours of operation

Links and Resources

Coming soon!

Contacts