Agilent SureGuide CRISPR Guide Libraries · 2017. 5. 12. · Building on Agilent’sLeadership in...
Transcript of Agilent SureGuide CRISPR Guide Libraries · 2017. 5. 12. · Building on Agilent’sLeadership in...
Agilent SureGuideCRISPR Guide Libraries
David Weiss
Field Application Scientist
Agilent Technologies
For Research Use Only. Not for use in diagnostic procedures.
Agilent’s Story"Innovating the HP Way"
Agilent’s Molecular expertise from enzymes to oligos
For Research Use Only. Not for use in diagnostic procedures.
Leading Provider of Measurement Tools & Solutions
Building on Agilent’s Leadership in Oligonucleotide Synthesis
Agilent’s DNA Oligo Library Synthesis (OLS) Manufacturing: SurePrint Technology• Longest and highest fidelity pooled oligos• Up to 230mer, up to 244K complexity pooled oligos
Mixture of pooled oligos
Release oligos from
surfaceCreate custom user-defined
oligo library design SurePrint oligo printing:Chemically synthesize olgioson array
DNA Oligo Synthesis RNA Oligo Synthesis
Agilent ConfidentialFor Research Use Only. Not for use in diagnostic procedures.
RNA
+
Protein
Clustered Regularly Interspaced Short Palindromic Repeats
Brouns lab website
Hsu et al., 2014
The complex is simple
8
dCA
S9
CAS
9
CAS
9
NHEJ(non
homologous end joining)
Homologous recombination CRISPR-A/I
For Research Use Only. Not for use in diagnostic procedures. Agilent Confidential
Enabling Life Sciences Researchin Complex Diseases
Genome sequencing has yielded 1000s of
disease associated candidate genes
Understanding the functions and roles of these
genes has been challenging
Cancer
Cardiovascular
Neurological
Metabolic
Autoimmune
Genetics Environment
Life Style
For Research Use Only. Not for use in diagnostic procedures.
13
Electrophoresis in Genome EditingCRISPR/Cas
January 11, 2016
For Research Use Only. Not for diagnostic procedures.
14
Fill-in reaction &
Purification
dsDNA template
In vitro gRNA transcription
DNase Digestion
DNA 1000 kit
gRNA purification
Purified gRNA
Small RNA kit
Small RNA kit
Minimal Design
Oligos
Extended Design forward
and reverse Primers
Synthesis of gRNA
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M2 -
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ntr
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Minimal Design ZFP42 Minimal Design GUSBControlExtended Design ZFP42Extended Design GUSBS
ize (
nt)
Average sizes of gRNA
Sizing precision:
• Guide RNAs were run in triplicate
• Highly precise (< 1% CV)
Agilent Application Note. 5991-7557EN
Electrophoresis in Genome EditingCRISPR/Cas
January 11, 2016
For Research Use Only. Not for diagnostic procedures.
15
Fill-in reaction &
Purification
dsDNA template
In vitro gRNA transcription
DNase Digestion
DNA 1000 kit
gRNA purification
Purified gRNA
Small RNA kit
Small RNA kit
Minimal Design
Oligos
Extended Design forward
and reverse Primers
Synthesis of gRNA
0.0200.0400.0600.0800.0
1000.01200.0
M1 -
ZFP
42
M2 -
ZFP
42
M3 -
ZFP
42
VM
1 -
ZFP
42
VM
2 -
ZFP
42
VM
3 -
ZFP
42
M1 -
GU
SB
M2 -
GU
SB
M3 -
GU
SB
VM
1 -
GU
SB
VM
2 -
GU
SB
VM
3 -
GU
SB
Co
ntr
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E1 -
ZFP
42
E2 -
ZFP
42
E3 -
ZFP
42
VE
1 -
ZFP
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VE
2 -
ZFP
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VE
3 -
ZFP
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E1 -
GU
SB
E2 -
GU
SB
E3 -
GU
SB
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GU
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Minimal Design ZFP42Minimal Design GUSBControlExtended Design ZFP42Extended Design GUSB
Quanti
ty (
pg
/µ
L)
Quantitation of gRNA
Yield Determination:
• Variable yield from IVT
• CV of < 13%
Agilent Application Note. 5991-7557EN
SureGuide in vitro offering
May 7, 2017
Confidentiality Label
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CAS
9
Purified CAS9 protein
purified guide RNA
SureGuide
gRNA
Synthesis
Kit
SureGuide
Cas9
nuclease
& controls
Programmable nuclease use in lieu of
Restriction Enzymes and PCR cloning
+
in vitro
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Agilent Application Note. 5991-7557EN
For Research Use Only. Not for use in diagnostic procedures.PR7000-0240
Catalog gRNA Libraries
Custom Human & Mouse gRNA
Libraries
Custom User Defined Libraries
Ready-to-Package Ready-to-Clone Ready-to-Amplify
• Plasmid Library
• GeCKOv2
• Human and Mouse
• Cloned into lentivirus
vector with hU6
promoter
• Pre-amplified oligo
library
• User defined subset
or designed
• Human and mouse
• Compatible with
SureVector cloning
• Unamplified oligo
pool
• Any species, any
cloning method
• Entirely custom by
user design
For Research Use Only. Not for use in diagnostic procedures.PR7000-0240
Catalog gRNA Libraries
Custom Human & Mouse gRNA
Libraries
Custom User Defined Libraries
Ready-to-Package Ready-to-Clone Ready-to-Amplify
• Plasmid Library
• GeCKOv2
• Human and Mouse
• Cloned into lentivirus
vector with hU6
promoter
• Pre-amplified oligo
library
• User defined subset
or designed
• Human and mouse
• Compatible with
SureVector cloning
• Unamplified oligo
pool
• Any species, any
cloning method
• Entirely custom by
user design
S
E
A
R
C
H
E
N
G
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Pooled
Arrayed
Discovery Using Pooled Guide RNA LibrariesSystematically find phenotypes associated with genes or a combination of genes
Pooled DNA Oligo gRNA Libraries
Introduce guide cassettes into host cell
Select for phenotype
• A faster, more efficient method for functional screening libraries
• Genes identified by selection for phenotype and sequencing of gRNA
• gRNA / Cas9 expression maintained through selection process
For Research Use Only. Not for use in diagnostic procedures.
For Research Use Only. Not for use in diagnostic procedures.
CRISPR Pooled Functional Screening Library Workflow
Lentiviral Delivery Workflow• Viral delivery can efficiently introduce
CRISPR sequences into cells
• Requires integration for expression
• Can be titered to Multiplicity of Infection
(MOI) of 1 or less
Lentiviral Preparation
For Research Use Only. Not for use in diagnostic procedures.
Representation
Customization
Fidelity
For Research Use Only. Not for use in diagnostic procedures.
The Agilent Advantage
Fidelity
Per cycle yields
during synthesis
CRISPR Libraries
Agilent
Competitor
Agilent
Competitor
• More correct constructs
• 92% vs. 77%
• Fewer avg. errors/kb
• 4 vs. 12
• Fewer errors with
longer constructs
• Improves library
quality
• Reduces screening
time and false
negatives
For Research Use Only. Not for use in diagnostic procedures.
The Agilent Advantage
Representation
Improved Representation
• Fewer missed
guides
• Less under-
represented
population
• Plasmid libraries
with 90/10 ratios
< 3
• Find all the hits in
your screen, not
just the over-
represented ones
Agilent
Competitor
Ag
ilen
t
Co
mp
etito
rs
For Research Use Only. Not for use in diagnostic procedures.
Modular Vector Assembly
- Each component is a
substitutable fragment
- Extensive set of standard
parts
- Use 1 reaction, and 1
system for all of your
cloning projects
- Amenable to automation
Agilent SureVector Chemistry: Advanced Library Cloning Method
Restriction/ligation methods
Type IIs restriction enzyme
based (Golden Gate)
Overlap annealing-based methods
Gibson assembly SureVector chemistry
Cut with Restriction enzyme
in the presence of ligase
and target vector
Ligated product
accumulates
Melt and anneal
Anneal, extend with polymerase and ligate
Recess ends with 5’-3’ exonuclease
Proprietary Enzyme mix
-Fast - Isothermal-Exonuclease optimization has large influence on efficiency
-Fast and most efficient-Standardized conditions-Repeat sequences can create muti-insertions
• Robust• Requires RE
Recognition Sequence
• -Lower efficiency
For Research Use Only. Not for use in diagnostic procedures.
Comparison of Gibson and SureVector cloned libraries
meas. hypoth. meas. hypoth. meas. hypoth.
missed guides 0 0 9 0 7 0
10th percentile 56 63 29 38 41 53
20th percentile 62 67 38 44 49 58
80th percentile 88 83 84 73 87 80
90th percentile 94 87 99 81 100 86
10/90 ratio 1.679 1.381 3.414 2.132 2.439 1.623
20/80 ratio 1.419 1.239 2.211 1.659 1.776 1.379
OLS Gibson SureClone
Missed Guides0.01%
10/90 ratio:2.5
For Research Use Only. Not for use in diagnostic procedures. Agilent Confidential
Plasmid Libraries: Transformation and Amplification
Assembly reaction
Transformation (electroporation)
Cleanup (SPRI beads)
Selection in very low melt
agar (2-3 days at 30 °C)
Harvest by centrifugation
Expansion in liquid
culture
For Research Use Only. Not for use in diagnostic procedures. Agilent Confidential
Customization
The Agilent Advantage
For Research Use Only. Not for use in diagnostic procedures.
Easy Customization
• Catalog quality,
pricing and lead-
times
• Any site, any
species, any
application
• Ready-to-clone
libraries for
mammalian system
(with design
service)
• Ready-to-amplify
libraries for total
flexibility
Catalog LibrariesHuman GeCKOv2 Mouse GeCKOv2
Part Number G7553A G7554A
Format Ready-to-package (plasmid
library)
Ready-to-package (plasmid
library)
Delivery Lentiviral (pSGLenti) Lentiviral (pSGLenti)
Targets Human exons Mouse exons
Application Genome-wide knock-out
screening
Genome-wide knock-out
screening
Content Pre-defined (no user design input) Pre-defined (no user design input)
Promoter U6 U6
Reporter GFP GFP
Selection Puromycin Puromycin
Sequence-verified plasmid library in pSGLenti vector
Ready-to-package into lenti-particles for mammalian cell delivery
For Research Use Only. Not for use in diagnostic procedures.
Custom Libraries (Ready-to-clone)Human/Mouse 10k Human/Mouse 30k Human/Mouse 60k
Part Number G7555A#010 G7555A#030 G7555A#060
Format Ready-to-clone (linear
DNA, pre-amplified)
Ready-to-clone (linear
DNA, pre-amplified)
Ready-to-clone (linear
DNA, pre-amplified)
Delivery Designed for lentivirus
(pSGLenti)
Designed for lentivirus
(pSGLenti)
Designed for lentivirus
(pSGLenti)
Targets User defined User defined User defined
Application Custom knock-out
screening, CRISPRa/i,
other CRISPR
applications
Custom knock-out
screening, CRISPRa/i,
other CRISPR applications
Custom knock-out
screening, CRISPRa/i,
other CRISPR
applications
Number of guides Up to 10,000 10,001-30,000 30,001-60,000
Content User defined (design
service available)
User defined (design
service available)
User defined (design
service available)
Compatible with SureVector library cloning kit (G7556A)
Cloning kit includes all reagents to generate an ultra-high quality plasmid library
For Research Use Only. Not for use in diagnostic procedures.
Custom Libraries (Ready-to-amplify)Custom 5k Custom 25k Custom 50k Custom 100k
Part number G7555V#005 G7555B#025 G7555B#050 G7555B#100
Format Ready-to-amplify
(oligo pool)
Ready-to-amplify
(oligo pool)
Ready-to-amplify
(oligo pool)
Ready-to-amplify
(oligo pool)
Delivery Any (user defined) Any (user defined) Any (user defined) Any (user defined)
Targets Any (user defined) Any (user defined) Any (user defined) Any (user defined)
Application Any (user defined) Any (user defined) Any (user defined) Any (user defined)
Number of guides Up to 5,000 5,001-25,000 25,001-50,000 50,001-100,000
Content Any (user defined) Any (user defined) Any (user defined) Any (user defined)
Available length Up to 200 bp Up to 200 bp Up to 200 bp Up to 200 bp
Includes amplification kit with all required reagents to generate a ready-to-clone library
For Research Use Only. Not for use in diagnostic procedures.
What matters in the end – what fraction of my library is correct?
average stdev average stdev average stdev
n
deletion across LTR 5.1% 2.3% NA NA
no error 87.8% 1.1% 92.6% 1.2% 91.1% 1.5%
point mutation 2.1% 0.4% 2.2% 0.4% 2.9% 1.1%
deletions 4.9% 0.8% 5.2% 0.9% 6.0% 1.3%
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plasmid libraries OLS
plasmid libraries OL
S
• ≈ 90% of all clones contain no detectable error
• ≈ 5% of clones contain a deletion
• ≈ 2% of clones may contain a point mutation
• For retroviral/lentiviral vectors the
recombination rate across the LTRs is ≈ 5%
Po
st-
pa
cka
gin
g
Pre
-pa
cka
gin
g
For Research Use Only. Not for use in diagnostic procedures.
Representation
Customization
Fidelity
For Research Use Only. Not for use in diagnostic procedures.
When pooled together it’s a fantastic pool
When pooled together it’s a fantastic pool
Representation
Customization
Fidelity
For Research Use Only. Not for use in diagnostic procedures.
Representation is key
to successful pooled
library screens.
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