UW-Madison Human Stem Cell Gene Editing Service
1
For each transgene or SNP correction, we will provide the following: • At least three positive clones • 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 UW-Madison Human Stem Cell Gene Editing Service A campus resource for applying CRISPR/Cas9 in human pluripotent stem cells Andrew Petersen, Ph.D.; Su-Chun Zhang, M.D., Ph.D.; and Anita Bhattacharyya, Ph.D. Who we are What we deliver Why we exist 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 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. Gene mutation correction/induction Endogenous site reporter insertion Lineage-tracing dual insertion Safe harbor site reporter insertion Acknowledgements Contact us A GlySer mutation (GC) in TDP-43 at A.A. 298 (G298S) causes the neurodegenerative disease Amyotrophic lateral sclerosis (ALS) WT TDP-43 G298S Using scar-free ssODN-CRISPR/Cas9 correction, we generated 7 clones of isogenic WT control cells TDP-43 G298G Off-target analysis on one clone revealed no mutations at 5 highest- predicted loci Email: [email protected] On the web: www.waisman.wisc.edu/cmn-gene-editing.htm 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. G.O.I. eGFP G.O.I. P2A loxP PuroR loxP PGK eGFP G.O.I. P2A loxP Cas9 D10A sgRNA (+ and – strand) sgRNA-F G.O.I. G.O.I. G.O.I. sgRNA-R =STOP G.O.I. P2A eGFP G.O.I. mCherry Fluorescent reporters Fluorescent fusions G.O.I. P2A Luciferase Luminescent reporters Protein tags G.O.I. FL AG HA + Puromycin CRE recombinase G.O.I. expression P2A self-cleavage G.O.I. eGFP Protein Tet-ON Safe-harbor locus insertion at the AAVS1 site allows over- expression or inducible expression of a construct of interest. Enzyme CAG promoter sgRNA-1 AAVS1 sgRNA-3 • Sustained open-chromatin status in most cell lineages • Pre-designed sgRNAs • Pre-designed “plug & play” donors • No CRE recombinase step – faster cell line completion Constitutive overexpression of protein Cell-type specific overexpression of protein eGFP Synapsin prom. Doxycycline-inducible expression (Dual construct insertion) Tet-TA CAG promoter T A Protein Tet-ON T A T A T A T A T A X No expression Protein The UW Human Stem Cell Gene Editing Service is supported by a UW2020:WARF Discovery Initiative grant and in part by a core grant to the Waisman Center from the National Institute of Child Health and Human Development (U54 HD090256). Su-Chun Zhang M.D., Ph.D. Co-director Anita Bhattacharyya Ph.D. Director Andrew Petersen Ph.D. Scientist 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. Endogenous locus CAG promoter 2272 mCherry 2272 loxP loxP AAVS locus CRE G.O.I. P2A G.O.I. CAG promoter 2272 mCherry loxP AAVS locus CAG promoter drives strong fluorescence, avoiding possible reporter intensity problems G.O.I. expression CRE-mediated recombination H9-AAVS-DIO-mCherry - TAT-CRE H9-AAVS-DIO-mCherry + TAT-CRE mCherry human Nuclei 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.
Transcript of UW-Madison Human Stem Cell Gene Editing Service
For each transgene or SNP correction, we will
provide the following:
• At least three positive clones
• 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
UW-Madison Human Stem Cell Gene Editing ServiceA campus resource for applying CRISPR/Cas9 in human pluripotent stem cells
Andrew Petersen, Ph.D.; Su-Chun Zhang, M.D., Ph.D.; and Anita Bhattacharyya, Ph.D.
Who we are What we deliverWhy we exist
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
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.
Gene mutation
correction/induction
Endogenous site
reporter insertion
Lineage-tracing
dual insertionSafe harbor site
reporter insertion
AcknowledgementsContact us
A GlySer mutation (GC) in TDP-43 at A.A. 298 (G298S) causes the
neurodegenerative disease Amyotrophic lateral sclerosis (ALS)
WT TDP-43 G298S
Using scar-free ssODN-CRISPR/Cas9 correction, we generated 7
clones of isogenic WT control cells
TDP-43 G298G
Off-target analysis on one clone revealed no mutations at 5 highest-
predicted loci
Email: [email protected]
On the web: www.waisman.wisc.edu/cmn-gene-editing.htm
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.
G.O.I.
eGFPG.O.I. P2A
loxP
PuroR
loxP
PGK
eGFPG.O.I. P2A
loxP
Cas9D10A
sgRNA (+ and – strand)
sgRNA-F
G.O.I.
G.O.I.
G.O.I.
sgRNA-R=STOP
G.O.I. P2A eGFP
G.O.I. mCherry
Fluorescent reporters
Fluorescent fusions
G.O.I. P2A Luciferase
Luminescent reporters
Protein tags
G.O.I. FL
AG
HA+ Puromycin
CRE
recombinase
G.O.I. expression
P2A self-cleavage
G.O.I. eGFP
ProteinTet-ON
Safe-harbor locus insertion at the AAVS1 site allows over-
expression or inducible expression of a construct of interest.
EnzymeCAG promoter
sgRNA-1
AAVS1
sgRNA-3
• Sustained open-chromatin status in
most cell lineages
• Pre-designed sgRNAs
• Pre-designed “plug & play” donors
• No CRE recombinase step – faster
cell line completion
Constitutive overexpression of
protein
Cell-type specific overexpression of
protein
eGFPSynapsin prom.
Doxycycline-inducible expression
(Dual construct insertion)
Tet-TACAG promoter
T
A
ProteinTet-ON
T
A
T
A
T
A
T
A
T
A
X No
expression
Protein
The UW Human Stem Cell Gene Editing Service is supported by a UW2020:WARF
Discovery Initiative grant and in part by a core grant to the Waisman Center from
the National Institute of Child Health and Human Development (U54 HD090256).
Su-Chun Zhang
M.D., Ph.D.
Co-director
Anita Bhattacharyya
Ph.D.
Director
Andrew Petersen
Ph.D.
Scientist
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.
Endogenous
locus
CAG promoter
2272
mCherry
2272 loxPloxP
AAVS
locus
CREG.O.I. P2A
G.O.I.
CAG promoter
2272
mCherry
loxP
AAVS
locus
CAG promoter drives strong fluorescence, avoiding possible
reporter intensity problems
G.O.I. expression
CRE-mediated recombination
H9-AAVS-DIO-mCherry
- TAT-CRE
H9-AAVS-DIO-mCherry
+ TAT-CRE
mC
he
rry
hu
ma
n N
uc
lei
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.
