A campus resource for applying CRISPR/Cas9 in in human pluripotent stem cells
The UW Human Stem Cell Editing Service, located in the Waisman Center provides CRISPR-Cas9 gene editing of human pluripotent stem cells (PSCs) to campus researchers.
Gene editing enables the creation of isogenic human PSC lines by correcting the gene mutation in the patient iPSCs or by knocking a mutation into otherwise normal human PSCs. The isogenic cells have the identical genetic background except for the mutation, thus providing stringent controls and avoiding much of the variability inherent in iPSCs, which will greatly enhance our ability to understand how those mutations cause particular diseases.
In addition to using gene editing to correct or establish mutations in hPSCs, the service will use new technologies to enable strategies to regulate gene expression, delete genes and insert tags that allow rapid cell sorting.
The gene editing sevice provides technical services, including generation of genome edited human stem cells lines, quality control of genome edited cells, and training for lab personnel.
About the Gene Editing Service
Who We Are
The UW–Madison Human Stem Cell Gene Editing Service, located at the Waisman Center and in conjunction with the iPS Core, will provide CRISPR/Cas9 gene editing of human pluripotent stem cells (hPSCs) to UW–Madison researchers.
Why We Exist
The development of CRISPR/Cas9 gene editing has altered how stem cell science is done. This technology allows quick and efficient generation of transgenic or isogenic lines for nearly unlimited research purposes that have previously been unfeasible. While we believe every researcher should have access to this technology, individual labs should not have to adopt the technology to reap the rewards. As a core service, we will serve as the experts in generating cell lines so labs can focus on addressing underlying scientific questions.
What We Deliver
For each transgene or SNP correction, we will provide the following:
- At least one positive clone
- At least one sequence–verified clone
- Off–target analysis for each guide RNA
- Mycoplasma testing (through WiCell)
- Karyotyping (through WiCell–extra charge)
- All available information for publication
- Method and protocol
- Sequence files (genomic, plasmid, primer)
- Off–target analysis results
Gene mutation correction/induction
Isogenic controls allow precise determination of an individual gene’s role in disease pathogenesis. Using either scar-free ssODN repair or plasmid-based repair, we can correct or induce various mutations.
A Gly→Ser mutation (G→C) in TDP-43 at A.A. 298 (G298S) causes the neurodegenerative disease Amyotrophic lateral sclerosis (ALS).
Off–target analysis on one clone revealed no mutations at 5 highest–predicted loci
Endogenous Site Reporter Insertion
Endogenous–locus insertion can report transcript presence and quantity, can facilitate drug screening or differentiation protocol optimization, or can generate protein tags for biochemical analysis.
Safe Harbor Site Reporter Insertion
Safe–harbor locus insertion at the AAVS1 site allows over–expression or inducible expression of a construct of interest.
Lineage-Tracing Dual Insertion
Lineage tracing uses 2 genetic insertions to stimulate sustained fluorescence in cells after a gene’s expression. We will generate “base” ES and iPS cell lines with the AAVS locus inserted for mCherry and eGFP.
This study was supported in part by a core grant to the Waisman Center from the National Institute of Child Health and Human Development (P50HD105353).