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Innovative CRISPR/Cas9 gene knockin and SNP-detection tools:
Elizabeth Quinn, PhD
Society for Laboratory Automation and Screening (SLAS)2018 conference
Establishing human iPS-derived disease
model lines for drug screening
Outline
• Introduction to genome editing and hiPSCs
• Knocking out genes in hiPSCs
– Key considerations
– Test case: generation of clonal cell lines with KO in CD81
• Knocking in genes in hiPSCs
– Key considerations
– Knockin of point mutations using ssDNA oligos (<200 bp)
– Knockin of longer sequences (>200 bp) with ssDNA repair template
2 © 2018 Takara Bio USA, Inc. All rights reserved.
Genome editing technologiesCRISPR/Cas9: bacterial mechanism of self-defense repurposed as an editing tool
3
Dominguez et al. Nat Rev Mol Cell Biol. 17, 5–15 (2016).
Cas9
endonuclease
sgRNA
Genomic DNACas9-sgRNA
complex
DNA cleavage+ Genome editing toolDNA target site recognition
Cas9 proteinsgRNA
© 2018 Takara Bio USA, Inc. All rights reserved.
Human iPSCs meet genome editingApplication of CRISPR/Cas9 in hiPSC-based disease modeling
4
Shi et al. Nat Reviews Drug Discovery 16, 115–130 (2017).
© 2018 Takara Bio USA, Inc. All rights reserved.
Knockout 5 © 2018 Takara Bio USA, Inc. All rights reserved.
Knocking out genes in hiPSCs
6
hiPS cells
Target gene
CRISPR/Cas9 Targeted gene knockout
Experimental design is essential in order to maximize success.
© 2018 Takara Bio USA, Inc. All rights reserved.
Knocking out genes in hiPSCsKey considerations
7
Design sgRNACas9-sgRNA
delivery into cells
(gene editing)
Single-cell
cloning
Screening
and validation
• Take into account different protein isoforms and alternative start codons
• Design sgRNAs targeting key exons or essential functional domains
• Use online tools to choose sgRNAs with predicted low off-target effects(http://chopchop.cbu.uib.no/index.php, https://www.deskgen.com/landing/)
• Check sgRNAs’ activity in vitro (Guide-it™ sgRNA Screening Kit)
• Use optimized sgRNA scaffold
© 2018 Takara Bio USA, Inc. All rights reserved.
Knocking out genes in hiPSCsKey considerations
8
Design sgRNACas9-sgRNA
delivery into cells
(gene editing)
Single-cell
cloning
Screening
and validation
• Minimize cell toxicity due to the delivery of the Cas9-sgRNA
• Delivery in the form of CRISPR/Cas9 ribonucleoprotein complex (RNP)
– Lowest toxicity in cells
– Relative transience: lower off-target effects
– No need for cellular transcription/translation machinery
Electroporation Gesicles
– No integration events: footprint-free genome editing
© 2018 Takara Bio USA, Inc. All rights reserved.
Guide-it rCas9 Electroporation Ready
Recombinant Cas9 purified from E. coli and ready for gene editing experiments using electroporation
– Sterile
– Contains one C-terminus Nuclear Localization Signal (NLS) protein
– Low glycerol content for higher electroporation efficiency/reduced toxicity
– Consistently effective when combined with Guide-it In Vitro sgRNA Transcription Kit
– Mix rCas9 and sgRNA, incubate for 5 minutes, and then use!
9 © 2018 Takara Bio USA, Inc. All rights reserved.
Guide-it rCas9 Electroporation ReadyKO of CD81 protein in hiPSCs
10
ChiPSC18
Edited population
© 2018 Takara Bio USA, Inc. All rights reserved.
Knocking out genes in hiPSCsKey considerations
11
Design sgRNACas9-sgRNA
delivery into cells
(gene editing)
Single-cell
cloning
Screening
and validation
• Minimize cell toxicity due to the delivery of the Cas9-sgRNA
• Delivery in the form of CRISPR/Cas9 ribonucleoprotein complex (RNP)
– Lowest toxicity in cells
– Relative transience: lower off-target effects
– No need for cellular transcription/translation machinery
Electroporation Gesicles
– No integration events: footprint-free genome editing
© 2018 Takara Bio USA, Inc. All rights reserved.
Knocking out genes in hiPSCsKey considerations
12
Editing machinery
design
Cas9-sgRNA
delivery into cells
(gene editing)
Single-cell
cloning
Screening
and validation
• Isolating and clonally expanding edited cells
– Single pluripotent cells die or differentiate when seeded alone
– Need for single-cell culture of pluripotent stem cells
© 2018 Takara Bio USA, Inc. All rights reserved.
Single-cell cloning of hiPSCs with the Cellartis® DEF-CS™ culture system
13
• Maintains cells in a highly undifferentiated state
• Allows for culturing iPS cells in a monolayer
• Feeder-free—no contamination, less time consuming, increased consistency
• Enables survival and expansion of single cells
• Maintains normal karyotype
• Allows rapid expansion for further downstream applications and analysis
iPS cell monolayer
© 2018 Takara Bio USA, Inc. All rights reserved.
Knocking out genes in hiPSCs Key considerations
14
Editing machinery
design
Cas9-sgRNA
delivery into cells
(gene editing)
Single-cell
cloning
Screening
and validation
• Characterization of the indels (Guide-it Indel Identification Kit)
• Check for nonexpression of your transcript by RT-PCR
Sharpe et al. Genome Biology 18, 109–113 (2017).
• Check pluripotency
• Check karyotype
© 2018 Takara Bio USA, Inc. All rights reserved.
Test case: knocking out CD81Workflow
Sorted
population
Single cell seeded
into each well of a
96-well plate
(by FACS sorting
or limiting dilution)
Expansion into
edited clonal
lines and
characterization
Editing of
hiPS cells to
KO CD81
15
FACS analysis
of the population
to determine
pluripotency and
% KO
© 2018 Takara Bio USA, Inc. All rights reserved.
ChiPSC22 or
ChiPSC18
Pluripotency maintained after CD81 KO
ChiPSC22 or
ChiPSC18
FACS analysis
of the population
to determine
pluripotency and
% KO
Editing of
hiPS cells to
KO CD81
0
20
40
60
80
100
Negative control Edited cells
ChiPSC18
0
10
20
30
40
50
60
70
80
90
100
Negative control Edited cells
ChiPSC22
% CD81+ % OCT-4+
% SSEA-4+
% p
ositiv
e c
ells
% p
ositiv
e c
ells
16 © 2018 Takara Bio USA, Inc. All rights reserved.
Serial dilution into 96-well plate
(~0.5 cells/well)
FACS into 96-well plate
Cloning of edited hiPSCs FACS or limiting dilution
17
Cell lineIsolation
method
Single
clones
Double
clones
Total clones
(proportion)
Total clones
(%)
ChiPSC18 FACS 52 0 52/96 54%
ChiPSC18Limiting
dilution46 12 58/55 105%*
*Percent expected versus total theoretical clones
© 2018 Takara Bio USA, Inc. All rights reserved.
Expansion of edited clonal lines
ChiPSC22 or
ChiPSC18
Sorted
population
Single cell seeded
into each well of a
96-well plate
(by FACS sorting or
limiting dilution)
Expansion into
edited clonal
lines and
characterization
FACS analysis
of the population
to determine
pluripotency and
% KO
Editing of
hiPS cells to
KO CD81
18 © 2018 Takara Bio USA, Inc. All rights reserved.
Edited, pluripotent single-cell clonesThe DEF-CS culture system maintains stemness
19 © 2018 Takara Bio USA, Inc. All rights reserved.
Edited, pluripotent single-cell clonesKaryotype analysis
20
Edited clonal cell line #3 Edited clonal cell line #5
© 2018 Takara Bio USA, Inc. All rights reserved.
Knockin21 © 2018 Takara Bio USA, Inc. All rights reserved.
Knocking in genes in hiPSCsKey considerations
22
Design sgRNA
and repair template
Cas9-sgRNA and
repair template
delivery into cells
Single-cell
cloning
Screening
and validation
• Check the sequence of the homologous recombination arms
• Pick sgRNAs as close as possible to the modification
© 2018 Takara Bio USA, Inc. All rights reserved.
Knocking in genes in hiPSCs Efficiency of SNP repair relies on close proximity to PAM site
23
Paquet et al. Nature 533, 125–129 (2016).
© 2018 Takara Bio USA, Inc. All rights reserved.
Knocking in genes in hiPSCsKey considerations
24
Design sgRNA
and repair template
Cas9-sgRNA and
repair template
delivery into cells
Single-cell
cloning
Screening
and validation
• Check the sequence of the homologous recombination arms
• Pick sgRNAs as close as possible to the DSB
• Use single-stranded donor templates
– No background expression when delivering expression cassettes (e.g.
CMVGFP)
– Lower rate of random integration than dsDNA
Chen et al. Nature Methods 8, 753-755 (2011)
© 2018 Takara Bio USA, Inc. All rights reserved.
Knocking in genes in hiPSCsRandom integration of dsDNA donors: GAPDH-AcGFP fusion in HEK293
25
0
2
4
6
8
10
negativecontrol
plasmid dsDNA ssDNA(S)
ssDNA(A)
- Cas9/sgRNA
+ Cas9/sgRNA
% flu
ore
scent cells
© 2018 Takara Bio USA, Inc. All rights reserved.
Knocking in genes in hiPSCsKey considerations
26
Design sgRNA
and repair template
Cas9-sgRNA and
repair template
delivery into cells
Single-cell
cloning
Screening
and validation
• Check the sequence of the homologous recombination arms
• Pick sgRNAs as close as possible to the DSB
• Use single-stranded donor templates
– No background expression when delivering expression cassettes (e.g.
CMVGFP)
– Lower rate of random integration than dsDNA
© 2018 Takara Bio USA, Inc. All rights reserved.
– Lower toxicity than dsDNA donors
Knocking in genes in hiPSCs ssDNA is less toxic than dsDNA
Cellular toxicity induced by dsDNA and ssDNA in hiPSCs
No donor template ssDNA dsDNA
– Cas9
+ sgRNA
+ Cas9
+ sgRNA
27 © 2018 Takara Bio USA, Inc. All rights reserved.
Knocking in genes in hiPSCsKey considerations
28
Design sgRNA
and repair template
Cas9-sgRNA and
repair template
delivery into cells
Single-cell
cloning
Screening
and validation
• Check the sequence of the homologous recombination arms
• Pick sgRNAs as close as possible to the DSB
• Use single-stranded donor templates
– No background expression when delivering expression cassettes (e.g.
CMVGFP)
– Lower rate of random integration than dsDNA
© 2018 Takara Bio USA, Inc. All rights reserved.
– Lower toxicity than dsDNA donors
– Repair mechanism more efficient than using dsDNA donorsRichardson et al. Biorxiv (2017).
Yan et al. Genome Res 27, 1099–1111 (2017).
Knocking in genes in hiPSCsKey considerations
29
Design sgRNA
and repair template
Cas9-sgRNA and
repair template
delivery into cells
Single-cell
cloning
Screening
and validation
• Bottleneck in homologous recombination experiments
• Homozygous vs. heterozygous
© 2018 Takara Bio USA, Inc. All rights reserved.
Knocking in genes in hiPSCs
30
Knockin
➢ Introducing a mutation
➢ Tag a protein
➢ Introduce a expression cassette in a safe harbor (like AAVS1)
© 2018 Takara Bio USA, Inc. All rights reserved.
Knocking in point mutations in hiPSCs Creation of isogenic cell lines
31 © 2018 Takara Bio USA, Inc. All rights reserved.
Homology-directed knockin of point mutationsUse of synthetic ssDNA oligos (<200 bp)
32 © 2018 Takara Bio USA, Inc. All rights reserved.
33 CONFIDENTIAL
Bottleneck in HR experiments: clone screening
SNP detection in
cell clones
(yes/no readout)
Mix of WT
Indels (NHEJ)
SNPs (HR)
KO SNP
N
Screening for SNP-containing clones using the SNP detection kit
34 PROPRIETARY AND CONFIDENTIAL | The Title of the Presentation
Workflow of SNP detection kit
Design and test displacement oligo and
flap probe
Extract genomic DNA of clonal cell lines
PCR amplification of the target sequence
Perform the enzymatic assay
Detection of all possible base mutations
35 © 2018 Takara Bio USA, Inc. All rights reserved.
Application in sample genotyping
36 © 2018 Takara Bio USA, Inc. All rights reserved.
Screening for SNPs related to tyrosinemia
37 © 2018 Takara Bio USA, Inc. All rights reserved.
KO SNP
N
Resolv
ase a
ssay
1 2 1 2 1 2 NC
sgRNA#1 sgRNA#2 sgRNA#3
16% 15% 0% 11% 18% 21%
1kb
ladder
1: RNP
2: RNP + HR donor
Screening for SNPs related to tyrosinemia
38 © 2018 Takara Bio USA, Inc. All rights reserved.
KO SNP
N
RF
LP
assa
yS
NP
de
tection k
it
Screening for SNPs related to tyrosinemia
39 © 2018 Takara Bio USA, Inc. All rights reserved.
Screening for SNPs related to tyrosinemia
41
Clo
nes
44
139
146
178
1810
10
20
30
40
50
60
70
80
90
100
41 44 139 146 178 181
FAH (p.Trp262Ter)
%OCT4 %TRA-160 % SSEA-4
% p
ositiv
e c
ells
40
Knocking in genes in hiPSCs
41
Knockin
➢ Introducing a mutation
➢ Tagging a protein
➢ Introducing a expression cassette in a safe harbor (like AAVS1)
© 2018 Takara Bio USA, Inc. All rights reserved.
Guide-it Long ssDNA Production SystemPCR-based method to create long ssDNA donors (>200 bp)
42 © 2018 Takara Bio USA, Inc. All rights reserved.
Guide-it Long ssDNA Production SystemPCR-based method to create long ssDNA donors (>200 bp)
43 © 2018 Takara Bio USA, Inc. All rights reserved.
PCR and “Strandase”-based preparation
– No cloning or gel purification
– So far, successful up to 5 kb
– Yield: 2–4 µg from 10 µg dsDNA
– Creating an ssDNA from a dsDNA PCR
product takes 30–40 minutes
Production of ssDNA ranging from 0.5–5 kb in length
44
CCR5 region
(0.5~5 kb)
Length
~0.5 kb
1 kb
2 kb
3 kb
4 kb
5 kb
ds ss ds ss ds ss ds ss ds ss ds ss ds ss ds ss ds ss ds ss
499 b 500 b 501 b 520 b 550 b 600 b 700 b 800 b 900 b 1000 b
ss ss ss ss100 b
pla
dder
1 k
bp
ladder
100 b
pla
dder
2 kb 3 kb 4 kb 5 kb
© 2018 Takara Bio USA, Inc. All rights reserved.
45
Knockin of EF1a-AcGFP1 at AAVS1 site
AcGFP1EF1aAAVS1
Isolated AcGFP1+
single cells
© 2018 Takara Bio USA, Inc. All rights reserved.
Isolated clonal lines without mutations
Left arm Right armAAVS1
locus
EF1a promoter AcGFP1 PolyA
L1
L2
R1
R2
L1/L2
R1/R2
NC #1 #2
Clonal cell lines
#3 #4 #5 #6 #7 #8 #9 #10 #11
46 © 2018 Takara Bio USA, Inc. All rights reserved.
Tagging of endogenous genes in hiPSCsN-terminal fusion of AcGFP1 to Tubulin
47
ssDNA (A) ssDNA (S)
ssDNA (AcGFP1-Tubulin)
Negative control
2.65% 1.30%
0%
Roberts et al. Biorxiv (2017).
© 2018 Takara Bio USA, Inc. All rights reserved.
Tagging of endogenous genes in hiPSCsN-terminal fusion of AcGFP to SEC16B
48
ssDNA (S) ssDNA (A)
ssDNA (AcGFP1-SEC16B)
Negative control
6.4% 7.6%
0%
Roberts et al. Biorxiv (2017).
© 2018 Takara Bio USA, Inc. All rights reserved.
Gact
Ctga
Knockin detection strategy
49
DNA
extraction
PCR
amplification
Single-cell
cloning
DNA
extraction
PCR
amplification
Displacement
oligo
WT vs. Knockin Flap probe
No signal
5’
3’
5’
3’
Non-edited
(G)
Edited
(knockin)
5’
3’
Guide-it FLAP
detector
Guide-it
Flapase
GCTT
CGAA
5’
3’
GACT
Ctga
5’
3’
Knockinn=2
CGAA3’
G5’3’T
5’
WT
© 2018 Takara Bio USA, Inc. All rights reserved.
Knockin detection strategy
Proof of concept
0
50
100
150
200
250
TubA1 SEC61B
wt FLAG tag NTC
Flu
ore
scence (
a.u
.)
50 © 2018 Takara Bio USA, Inc. All rights reserved.
Takara Bio and stem cell expertise
51
Takara Bio USA, Inc.
Mountain View, CA
Takara Bio
Takara Bio Europe AB
Gothenburg, Sweden
Takara Bio Inc.Kusatsu, Shiga, Japan
Production & Development
• Sales & Distribution– Takara Bio Europe S.A.S.
– Takara Bio USA, Inc.
– Takara Biomedical Technology Co. Ltd.
– Takara Korea Biomedical Inc. DSS
– Takara Bio India Pvt. Ltd.
© 2018 Takara Bio USA, Inc. All rights reserved.
Cellartis products and services
Undifferentiated human pluripotent stem (hPS) cells– hPSC lines
– DEF-CS and DEF-CS Xeno-Free (2D, 3D, and GMP) for expansion and maintenance
– Media kits for gene editing and single-cell cloning
– Differentiation kits
Specialized cells, differentiated from hPS cells– Definitive endoderm cells
– Hepatocytes
– Beta cells
– Cardiomyocytes
Human pluripotent stem cell services– Sourcing, reprogramming, and banking
– Directed differentiation into multiple cell types
– Clinical-grade human embryonic stem cell line generation and banking
– Gene editing
Miscellaneous– Power™ Primary HEP Medium for primary hepatocytes
– Adult neural stem cells
– Medium for expansion, maintenance, and differentiation of neural stem cells
– Antibodies and qPCR primers for characterization and detection
52 © 2018 Takara Bio USA, Inc. All rights reserved.
THANKS!
53 © 2018 Takara Bio USA, Inc. All rights reserved.