Advancements in Genome Editing, Gene Synthesis, and Engineered Cell Models

Advancements in Genome Editing, Gene
Synthesis, and Engineered Cell Models
University of Manitoba
October 5th, 2015
Synthetic Biology Technical Sales Specialist
Jennifer Blake
[email protected]
The world leader in serving science
1
Gene Synthesis, Genome Editing Tools &
Solutions Outline
• Background
• Gene synthesis and Strings
• Genome engineering
• Workflow overview
• CRISPR-Cas9 technology: DNA, mRNA, Protein
• In depth on Cas9 protein
• Design
• gRNA and donor plasmids
• Delivery
• Detection and Selection Tools
• Genomic Cleavage Detection Assay
• Engineered Cell Models
2
About us
Access to the most comprehensive and trusted molecular biology reagents and services
Shaping discovery. Improving life.
• We believe in the power of science to transform lives. To support scientists
worldwide, we offer high-quality, innovative life science solutions—from everyday
essentials to instruments—for every lab, every application.
Carlsbad, CA
3
Regensburg, Germany
Pleasanton, CA
Opportunities offered by Gene Synthesis
GeneArt® Gene Synthesis increases quality, reliability and success rate
 cost-effective, time- and resource-saving method for
 obtaining individual and desired DNA constructs
 with 100% sequence accuracy
 providing equivalent or better expression performance.
4
Paralleled approaches
High throughput
High throughput
Automated
Automated CE sequencing
Automated alignment
Product report
Shipment
ISO 9001
5
LIMS
Nano scale synthesis
High throughput/quality
High Throuput Process
Optimization/CAD/Portal
Design fragments and oligos
Alignment and simulation in
silico
Specialized or Large Projects
Gene Synthesis: Technologies and Processes
Individual anaylsis and
design
Project handling
through PMO
Deployment of HTP
production for raw
materials
Complex or
individualized assembly
technologies
Special manufacturing
unit for extended
process requirements
Tight communication
patterns
Individual technical
responses
Product report
Shipment
ISO 9001
GeneArt® Gene Synthesis Production Times
Faster times coming soon
Gene Length
Processing time (business days)
Standard 1)
≤1,200 bp 2)
Express 1)
SuperSpeed 3)
9
7
5
1,201 2) - 1,800 bp
14
12
7
1,801 - 3,000 bp
14
12
n/a
3,001 - 5,000 bp
19
17
n/a
> 5,000 bp
Get a quote
Get a quote
n/a
(SuperSPEED 1.2)
(SuperSPEED 1.8)
For complex sequences: Get a quote
1) Valid for standard gene synthesis (non-complex, GC content 10% - 80%).
2) In case of GC content of 10% - 20% or 75% - 80%: 1,000 bp instead of 1,200 bp.
3) Subject to sequence assessment. Order must be placed by 3:00 p.m. CET.
Please note: Rarely, requested sequences are found to be toxic and/or genetically unstable. These production times are only valid for
nontoxic sequences that are genetically stable in E. coli.
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Vectors for Express
• pcDNA3.1(+)
Mammalian
• pcDNA3.3-TOPO
• pcDNA3.4-TOPO
Insect
• pFastBac1
• pET100/D-TOPO
• pET151/D-TOPO
Bacterial
• pRSET A
• pYes2.1V5-His TOPO Yeast
Gateway
• pDONR221 NEW
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GeneArt® Strings™ DNA Fragments 0.1-3.0kb
synth error
Enzymatic
error correction
+ PCR amplif.
oligo
assembly
Strings™ 0.1-1kb
mutation
Conc. Determ.
QC (bulk sequencing)
Dry down
Shipment
Enzymatic error
correction
Fragment assembly &
amplification by
Fusion PCR
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Strings™ 1-3kb
GeneArt® Strings™ DNA Fragments Pricing
Strings <1kb
Strings 1-3kb
(HARPO)
9
Length (bp)
Price CAD
100 to 600
$
81.75
601 to 750
$
104.25
751 to 1000
$
127.20
1001 to 1250
$
215.10
1251 to 1500
$
251.10
1501 to 1750
$
287.10
1751 to 2000
$
332.10
2001 to 2250
$
377.10
2251 to 2500
$
431.10
2501 to 2750
$
476.10
2751 to 3000
$
521.10
Genome editing work-flow
Rapid and efficient editing with multiplexing capabilities
The world leader in serving science
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What is genome editing?
• Genome editing is an approach whereby a genomic DNA sequence is directly changed
by adding, replacing, or removing DNA bases.
Removing DNA
Adding DNA
Diseaseresistant
transgenic
plants
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Gene
therapy
Replacing DNA
Animal disease
models
Stem cell
engineering
Tissue disease
models
• To study gene function
• To label endogenous genes
• To target gene mutation
• Stable integration
• To target transgene addition for heritable
modification
• For tissue & cell engineering to produce novel functions
Genome editing with designer engineered nucleases
Traditional methods
Cell’s repair mechanism is
harnessed to heal DNA breaks
12
Modern/emerging
Genome/cell engineering workflow
With engineered nucleases
13
Step 1: Design
Genome/cell engineering workflow with engineered nucleases
Design
Identify target sites, design &
order
Deliver
Detect
Transfect
Screen, enrich & validate
Online Design Tools
Online Design Tools
• lifetechnologies.com/TALs
• lifetechnologies.com/CRISPR
Genome Editing Tools
• GeneArt™ TAL nucleases
• GeneArt™ CRISPR nuclease
• GeneArt™ Cas9 mRNA
• GeneArt™ Cas9 protein
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GeneArt TALs
Step 2: Deliver
Genome/cell engineering workflow with engineered nucleases
Design
Deliver
Detect
Transfect
Screen, enrich & validate
Identify target sites, design &
order
•
Gibco™ cell culture media
•
Transfection reagents for DNA & RNA
• Lipofectamine™ 3000 Transfection Reagent
• Lipofectamine™ 2000 Transfection Reagent
• Lipofectamine™ MessengerMAX™ Transfection
Reagent
•
Neon™ Transfection System
•
Cell imaging reagents and tools
Gibco products
Neon Transfection System
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Lipofectamine 3000
Transfection Reagent
EVOS™ FL Imaging System
Step 3: Detect
Genome/cell engineering workflow with engineered nucleases
Design
Identify target sites, design &
order
•
•
•
•
Deliver
Detect
Transfect
Screen, enrich & validate
GeneArt Genomic Cleavage Detection Kit
GeneArt Genomic Cleavage Selection Kit
TaqMan™ SNP Genotyping Assay
Ion PGM™ sequencing
GeneArt Genomic Cleavage Detection Kit
GeneArt Genomic Cleavage Selection Kit
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QuantStudio™ 7 Flex
Real-Time PCR System
Ion PGM System
GeneArt Genomic Detection Kit
Simple, reliable, and rapid method for detection of
locus-specific gene modification
The world leader in serving science
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GeneArt Genomic Cleavage Detection Kit
An essential tool for monitoring the efficiency of your genome editing
• To detect cleavage activity of engineered nucleases on endogenous target loci
• Easy—no genomic DNA isolation, direct PCR amplification
• Rapid— ~5hour total processing time
• Quantative—gel band density directly correlated to target indel
introduction
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Efficient PCR amplification of genomic locus
From cell lysates of various cell types
Direct PCR amplification of target locus from cell lysates
Lysis
Numbers of cells
PCR
M
M
Effectively lyse 1x104 to 2x106 cells in 50 μL of lysis buffer
Numbers of cells (x106) M
HeLa
Hepa*
HepG2
HT29
A549
1
1
1
1
1
2
2
2
*Hepa is mouse cell line used as negative control
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2
2
M
CRISPR-Cas9 genome editing
Rapid and efficient editing with multiplexing capabilities
The world leader in serving science
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The CRISPR-Cas9 revolution
How it works?
1.
2.
3.
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Cas9 is a bacterial nuclease
Small RNA (“guide RNA”) directs Cas9 to the
site of cleavage
Cas9 can be targeted to any genomic locus of
interest to introduce DNA double strand break
Available CRISPR-Cas9 delivery formats
The choice is yours! Complete suite of tools & reagents
NEW!
NEW!
Lentiviral
CRISPR-Cas9
IVT gRNA
+
Cas9 Protein
Lentiviral gRNA
GeneArt® CRISPR all-in-one
GeneArt® CRISPR
GeneArt® CRISPR
Plasmid
mRNA
GeneArt® CRISPR
Protein
Lentiviral Libraries
Cell transfection



•
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Multiplexing gene targeting
Microinjection
HTP Genome Engineering
•
Loss-of-function screening
1) GeneArt® CRISPR nuclease vectors
All-in-one reporter vector system
• A simple ready-to-use, all-in-one expression vector system consisting of
both a Cas9 nuclease expression cassette and a guide RNA cloning
cassette for rapid and efficient cloning of a target-specific crRNA
GTTTT
GTGGC
Target-specific
custom oligo
GeneArt®
CRISPR
Nuclease-CD4
vector
9,822 bp
CD4 vector: For bead-based enrichment of
Cas9+gRNA– expressing cells
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GeneArt®
CRISPR
Nuclease-OFP
vector
9,219 bp
OFP vector: For fluorescence-based
enrichment of Cas9+gRNA–expressing cells
GeneArt® CRISPR Nuclease mRNA and protein
GeneArt® CRISPR U6 Strings™ DNA
gRNA expression cassette with U6 Pol II promoter
GeneArt® CRISPR
Nuclease mRNA
GeneArt® CRISPR
Nuclease Protein
+
or
GeneArt® CRISPR T7 Strings™ DNA
(in vitro transcribed gRNAs)
IVT gRNA generated from DNA template containing
T7 promoter
Advantages
• No promoter constraint for Cas9 expression
• Smaller payload size (U6 CRISPR String™
=500 bp & IVT gRNA =100 bp)
• Allows Cas9 to gRNA dosage optimization
• Amenable to multiplexing
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Comparing CRISPR/CAS9 Delivery Formats
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GeneArt® CRISPR-Cas9 Protein
Rapid and efficient editing with multiplexing capabilities
The world leader in serving science
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Thermo Fisher’s Genome Engineering Goals
Allow both new and experienced scientists easy access to transformative genome
editing tools by enabling:
‒
‒
‒
‒
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Simple methods for design of CRISPR tools
Easy ways to order CRISPR tools
Simple to use CRISPR tools
High efficiency specific tools which minimize downstream screening
Cas9 Protein Workflow
Design to Cleavage analysis in 4 days
Coming Oct 15th: Lipofectamine CRISPRMax
Advantages of Cas9 RNP mediated genome editing:
• Ready to act once delivered into the cell (no transcription or translation need to make the protein)
• No foot print left
• Controlled Dose. Efficient for both single and multiplexing
• Stable
• Fast Turnover
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GeneArt™ Precision gRNA Synthesis Kit
• Simple, quick & robust gRNA synthesis
• Design -> delivery and cleavage
efficiency data by GCD within 5 days
• Web tool for oligo design & purchase
• Worfklow:
1.
2.
3.
4.
5.
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Design target specific oligos with
CRISPR Search & Design webtool
Combine tracer/oligos, target specific
oligos, Phusion Master Mix & dNTPs
Perform PCR
Perform in vitro transcription & clean up
Result: gRNA in RNA format ready for
complex with protein
Time Course of Cas9 Activity in 293FT
Time course of cleavage
0
4
8
Western Blot
0
24 48 72 hrs
DNA
DNA
%cut: 0
0
4
42 57 68
mRNA
mRNA
%cut: 0
0
8
50 62 68
protein
Protein
%cut: 0
0
4
48 64 70
24 well, DNA 1 ug, Cas9 mRNA 500 ng/IVT gRNA 50 ng,
Cas9 protein 500 ng/IVT gRNA 50 ng
Used a normalized dosage of plasmid, mRNA or protein to get
the same level of cleavage for this comparison
30
Liang et al. in press
4
8
24 48 72 hrs
Comparison of Off-target effect using plasmid, mRNA,
and protein formats
On T3
GGTGAGTGAGTGTGTGCGTGTGG
OT3-T2 AGTGAGTGAGTGTGTGTGTGGGG
OT3-T18 TGTGGGTGAGTGTGTGCGTGAGG
The off-target sites of VEGF were identified by
GUIDE-seq from Keith Joung’s group (same
dosages)
Lipid-mediated Transfection in 293FT
mRNA and protein have higher on target and lower off target than plasmid
possibly due to the lower maintained levels of Cas9 in the system
31
Liang et al. in press
Electroporation-mediated transfection of iPSC
(C) Flow cytometry
(A) 24 optimized protocol
Tra -1-60- AlexaFluor®488
Unstained
Pulse, voltage, # of pulses, and pulse
width
Cas9 mRNA
mRNA
Control
Cas9 Protein
Cas9
(B) Transfection via Electroporation
ug protein
Neg DNA mRNA 1
2
3
SSEA4-AlexaFluor®647
% Cut:
32
0
20
31
62
80
Liang et al. in press
89
93% dual positive in control vs 85% after
transfection
Multiplex Knockout with Cas9 in Jurkat T (Male)
(A) AAVS, RelA, and HPRT targets
Neg
(B) Efficiency of triple knockout
Protein
1
AAVS
% Cut:
0
76
79
RelA
% Cut:
0
81
88
Zygosity
0.8
0.6
0.4
0.2
0
HPRT
% Cut:
33
0
91
90
Liang et al. in press
AAVS
RelA
HPRT
All
1 ug Cas9 protein, 200 ng gRNA, 1x10^5 cell in 24 well
3 genes (5 alleles since HPRT is on the x chromosome and the Jurkat T
is male)
1 ug Cas9 protein, 200 ng gRNA, 1x10^5 cell in 24 well
3 genes (5 alleles since HPRT is on the x chromosome and the Jurkat T
is male)
+/+
-/+/-
Transfection efficiency in variety of cell line
Overall best results with Cas9 RNPs
Comparison of plasmid DNA, Cas9 mRNA/gRNA, and Platinum
Cas9 RNP transfection and editing efficiencies in variety of cell lines
Plasmid
Cell Lines
mRNA
To be updated with
new mRNA data,
Lipofectamine®
CRISPRMAX™
Cas9 Transfection
Kit & more cell types
for marketing image
Protein
Lipid
Electro
Lipid
Electro
Lipid
Electro
HEK293FT
49
49
70
40
51
88
U2OS
15
50
21
24#
18
70
Mouse ESCs
30
45
45
20
25
70
Human ESCs (H9)
0
8
20
50
0
64
Human iPSCs
0
20
66
32
5
87*
N2A
66
75
66
80
66
82
Jurkat
0
63
0
42
0
94*
K562
0
45
0
27
0
72
A549
15
44
23
29
20
66
Human Keratinocytes (NHEK)
0
30
0
50
0
35
n/a
0
n/a
0
n/a
24
Human Cord Blood Cells CD34+
34
HPRT for human cell lines and Rosa 26 for mouse cell lines
* Confirmed by sequencing
GeneArt™ CRISPR Nuclease mRNA: new sku
Improved manufacturing process for improved performance
A) Gel picture on Bioanalyzer
B) Electropherogram on Bioanalyzer
Cas9 mRNA
Cat# A29378
Cas9 mRNA Cat# A29378
Cat# A29378
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Cas9 mRNA Cat# A29378
1) 500ng Cas9 mRNA was cotransfected with 100ng
HPRT T1 gRNA IVT to
each well cells in 24-well
plate format.
2) Cells were harvested after
72 hours post-transfection.
3) GCD assay for HPRT locus
were performed and
function of Cas9 mRNA
was determined by
cleavage efficiency based
on band intensity.
GeneArt™ CRISPR Search
and Design Tool
The world leader in serving science
36
Enter Search Parameters
https://www.lifetechnologies.com/CRISPRDesign
37
Search Results
38
Predicting binding sites
https://www.lifetechnologies.com/CRISPRDesign
39
Donor DNA design and synthesis included
Genomic locus
Donor DNA
•
•
•
•
•
40
Right homology
Left homology
Region intended
for change
Length: Optimal length of homology: 0.5 - 1 kb
Position: Place region to change in the middle of the donor, at least 500 bases of homologous
sequences flanking
Stability: Strategies may be adopted to make donor DNA resistant to the cut by the engineered
nuclease (eg. silent mutations in CRISPR/TALN binding site)
Formats:
• Plasmid
• Linearized plasmid
• Excised from plasmid
• PCR product (from genome or plasmid)
• ssDNA
Intended changes:
• Single nucleotide polymorphism (introduce or revert a SNP)
• Knock-in of an exogenous gene into a “safe harbor” (eg. AAVS1, HPRT, mROSA26)
• Gene disruption using an antibiotic marker
• Introduction of reporter (eg. fluorescent fusion protein, promoter trap)
Confirmation of gene editing by Sanger
sequencing
Target HPRT in iPS cells
Target HPRT in Jurkat cells
gRNA
Ctrl DNA
mRNA
Protein
Cas9
Cleavage efficiency%
Clone amplified
PCR product into
TOPO® TA vector
94
Transform into One Shot®
TOP10 chemically
competent E. coli cells
Gene modification efficiency: 94.1%
41
0 20
Cleavage efficiency
detected by GCD assay
correlated with results by
Sanger sequencing
32 62 81 89
Isolate DNA using
PureLink® Pro 96
Purification Kit
Gene modification efficiency: 87.8%
Sanger sequencing using
3500xL Genetic Analyzer
Sequence analysis using
Vector NTI® Software
GeneArt™ Engineered Cell Models
The world leader in serving science
42
What is it?
GeneArt™ Engineered Cell Models
The world’s largest collection of ready-to-go CRISPR engineered cell lines
• Engineered cell lines starting at less than $1,000
• 1,000s of off-the-shelf knockout and knock-in cell
models (5-7 bd for off the shelf)
• Affordable, rapid production of made to order cell
lines (10 weeks)
• All models ship with the matched isogenic parental
cell line
43
GeneArt™ Engineered Cell Models
Knockout cell lines
•
•
44
Complete gene knockouts
Knockout cell lines can be haploid or
diploid
Knock-in cell lines
•
Have more complex genomic
modifications (e.g. deletions, single
base pair changes, insertions)
•
Knockin cell lines are diploid
•
Custom knockout cell line generation
is available on demand
•
•
Custom knockin cell line generation is
available on demand
Over 1,500 cell lines and growing
•
500 cell lines covering >350
genotypes
Available gene sets
Gene sets coming soon:
• Phosphoinositide Metabolism
• Protein phosphatases
• Glycosylation disorders
• TRIM E3 ligases
• Suggestions??
Which genes are available?
•
•
•
45
Knockouts for more than 1,400 human genes in stock
Various gene sets: Kinases, Epigenetics, Ubiquitination, Signaling
Customer can either purchase individual knockout cell lines or entire sets
Gene family collections
Gene Collections
•
•
•
•
46
Knockouts for entire gene families
Comprehensive: Coverage of all non-essential genes that are
expressed in HAP1
Several clones per gene available
Kinases, Bromodomain genes, Deubiquitinases, Ubiquitin E2
ligases, HDACs, Caspases, Rab GTPases
Pathway collections
CMAS
SLC35A1
Fluorescein
SNA Lectin
Brightfield
HAP1 wt
Pathway Collections
•
Knockouts for entire biological pathways
•
Allows comprehensive study of processes
•
Several clones per gene available
•
Sialylation, mTOR signaling, TNF- signaling, Autophagy,
Epigenetics, DNA damage responses
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Ordering Information
• Fast, simple web ordering
tool with a built-in search &
add to cart functionality
• How to order
① Select institution type
② Enter in either
①
Gene name
②
or Gene ID
③ Select cell line
④ Add to cart
⑤ Check out
48
Production pipeline
Shipment
Packagin
g
Quality Control
49
Customer
Custom knockouts
for any human gene
in 10 weeks
Design
Production
Pricing CAD
Customer
Segment
Academic
-
Off-the-shelf cell
lines
Made-to-order
knockout
HAP1 cell lines
Made-to-order
knockin
cell lines
$1,287
$2,574
$4,420
Bulk discounts are available
Gibco media can be bundled
Knock out is guaranteed
14,000 non-essential knock outs available in Hap1
Made to order knockout and knock-in models available Q4 2015
50
Recommended reagents for Hap1 cell lines
51
Workflow needs and solutions
A collection of optimized and validated end-to-end solutions
Thermo Fisher Scientific, a leading innovator in genome engineering,
provides tools and solutions for the complete gene engineering workflow.
Visit www.lifetechnologies.com/cellengineering for more information
52
Questions & Answers
The world leader in serving science
53
For Research Use Only. Not for use in diagnostic procedures.
© 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a
registered trademark of Roche Molecular Systems, Inc., used under permission and license. TALEN is a trademark of Cellectis.
54
Appendix
Additional information
The world leader in serving science
55
Choosing the right designer nuclease for your research
RNAi
CRISPR
TALs
Programmable RNA-guided DNA
endonuclease
DNA-binding domain to recognize
virtually any sequence
Key
capabilities
•
Gene knock-down
•
•
•
Gene knock-out
Gene knock-down
Gene knock-in
•
•
•
•
Gene knock-out
Gene knock-down
Gene knock-in
Gene activation
Target
customers
•
•
•
•
High-content imaging groups
System biology
Disease-focused labs
RNA-Seq groups
•
•
•
•
•
•
•
High-content imaging groups
System biology
Disease-focused labs
RNA-Seq groups
Metabolic engineering
Protein production
Genome engineering cores
•
•
•
•
•
•
•
High-content imaging groups
System biology
Disease-focused labs
RNA-Seq groups
Metabolic engineering
Protein production
Genome engineering cores
Price/rxn
$200/rxn
$50/rxn
$2000/rxn
Off-target
effects
Unpredictable
Possible
Few
Why choose
this option?
Transient knock-down of RNA
transcript(s) for multiple targets
Great for screening, provides rapid
editing with multiplexing capabilities
Precise editing when you want to
target a single gene, activate a gene
of interest, or working with
nonmammalian cell types
Discovery to validation
56
When to choose TALs vs. CRISPRs
Will use edited cells for commercial applications (need clear IP
landscape)
TALs
TAL
mRNA
✔
✔
CRISPR
vector
CRISPR
mRNA
Multiplexing (edit multiple sites at once)
✔
Screen multiple sequences at once for optimal sequence ID
✔
✔
Microinjection (in vivo applications)
Minimal off-target editing
Larger
footprint
Need to remove promoter constraints and express in a broad
range of cell types
✔
Larger
footprint
✔
✔
No sequence constraint (for example, no NGG PAM restriction)
✔
Gene repression (knock-down)
✔
Gene knock-out
✔
✔
✔
✔
Gene knock-in
Service
Service
Service
Service
Gene activation
✔
Need to edit non-mammalian cell types
✔
*
*
Custom design included
✔
✔
✔
✔
Cell engineering services
✔
✔
✔
✔
*These are mammalian-optimized systems; however, they have been shown to work in nonmammalian cells. For host57 specific optimization, please contact [email protected]
Arrayed and pooled library screening
Screening of arrayed library
 4 gRNAs per gene
 > 106 TU/mL
One gene per well
 Much more controlled delivery of gRNA per well
 Eliminate time-consuming deconvolution step
 Require some level of automation
Screening of pooled ;ibrary
 10 gRNAs per gene
 108 TU/mL
Multiple genes in parallel
 Much less expensive than arrayed format
 Do not require special infrastructure
Focused gene; libraries: Kinases, Phosphatases, GPCR
58