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: geneediting@waisman.wisc.edu

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.