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

0

20

40

60

80

100

M1 -

ZFP

42

M2 -

ZFP

42

M3 -

ZFP

42

VM

1 -

ZFP

42

VM

2 -

ZFP

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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

ol

E1 -

ZFP

42

E2 -

ZFP

42

E3 -

ZFP

42

VE

1 -

ZFP

42

VE

2 -

ZFP

42

VE

3 -

ZFP

42

E1 -

GU

SB

E2 -

GU

SB

E3 -

GU

SB

VE

1 -

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SB

VE

2 -

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SB

VE

3 -

GU

SB

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

ol

E1 -

ZFP

42

E2 -

ZFP

42

E3 -

ZFP

42

VE

1 -

ZFP

42

VE

2 -

ZFP

42

VE

3 -

ZFP

42

E1 -

GU

SB

E2 -

GU

SB

E3 -

GU

SB

VE

1 -

GU

SB

VE

2 -

GU

SB

VE

3 -

GU

SB

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

16

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

17

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

I

N

E

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%

44 10

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.

CRISPR Promo

30% OffAgilent’s CRISPR BioReagents

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Sanju.Sharma@non.agilent.com

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Liz_Leigh@agilent.com