New Tools for Mammalian GenomeNew Tools for Mammalian Genome Engineering with the CRISPR‐Cas9 System

Fangting Wu

9/26/20139/26/2013

1

OverviewOverview

• Introduction of CRISPR/Cas system/ y

• SBI’s PrecisionX TM Cas9 System

All‐in‐one vector systemAll‐in‐one vector system

RNA system

G diti i P i i X TM C 9• Genome editing using PrecisionX TM Cas9 All‐in‐one or RNA system

Q&A• Q&A

CRISPR‐Cas as a new tool for genome i iengineering

Science 2013;339 (6121):768-770

What is CRISPR‐Cas?What is CRISPR Cas?

( l d l l d h• CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

• Cas (CRSPR‐associated proteins)( p )

CRISPR‐Cas system in prokaryoteCRISPR Cas system in prokaryote

CRISPR‐Cas system in prokaryote

CRISPR‐Cas9 system as a genetic editingtool in bacteriatool in bacteria

crRNA maturation

(NGG)

Jinek et al Science 2012;Jinek et al. Science 2012; 337 (6096):816-821

CRISPR‐Cas9 system as a genetic editingtool in bacteria

Cas9 vCI

CII

CIII

HNHTarget DNA

Cas9

tool in bacteria

Cas9

Ruv

Ruv

RuvCHNH

gRNA

Cas9 (D10A) Nickase

RuvC

I

RuvC

II

RuvC

III

HNHTarget DNA

gRNA

Cas9 (D10A)

C 9 (H840A) Ni k

I II II Target DNA

g

Cas9 DM

Cas9 (H840A) Nickase

RuvC

RuvC

I

RuvC

IHNH

g

gRNA

CRISPR‐Cas9 system as a genetic editingtool in bacteriatool in bacteria

PAM (NGG)

CRISPR‐Cas9 system as a genetic editingl i h lltool in human cells

Mali et al. Science. 2013, 339:823-6

CRISPR‐Cas9 system as genetic editing y g gtool in different model of organisms

CRISPR‐Cas9: more efficient than TALEN

Ding et al. Cell Stem Cell. 2013 12(4):393-4

T ti ifi it f CRISPR C 9Targeting specificity of CRISPR‐Cas9

Paired Nickases enhance genome editing specificity

Gene regulation by catalytically g y y yinactive Cas9 DM

Cas9 DM

RuvC

I

RuvC

II

RuvC

III

HNHTarget DNA

gRNA

Cas9 DM

SBI PrecisionX TM Cas9 SystemSBI PrecisionX Cas9 System

SBI PrecisionX TM Cas9 SystemSBI PrecisionX Cas9 System

All‐in‐one RNAAll‐in‐one vector system

RNA system

hspCas9 SmartNuclease

hspCas9 (D10A) SmartNickase

hspCas9 DM      NullNuclease Cas9 mRNA T7 gRNA kit

hspCas9 mRNA

hspCas9 (D10A) mRNA

hspCas9 DM mRNA

spCas9 mRNA (prokaryote)

ApplicationsApplications

• Genome editing and engineering of modelGenome editing and engineering of model organisms

• Gene correction for disease specific iPSC• Gene correction for disease specific iPSC

• Synthetic biology applications

• Gene/Cell‐based therapy

SBI PrecisionX TM Cas9 System comparisonSBI PrecisionX Cas9 System comparison  

All‐in‐one vector system RNA system

Genome engineering in  Transgenic animal 

model for in vivomammalian cell lines

model for in vivo , ex vivo study

Genome modification in ES 

cells or iPSCs

Cas9 products comparison ChartFunction Targeting

effectOn target efficiency

HDR efficiency

Off target indels

Cas9 products comparison Chart

Cas9 SmartNuclease

High efficient genome editing by NHEJ or HDR

Double‐strandedDNA breaks

high high Yes

Single Cas9SmartNickase

Genome modification by HDR

Single‐strandedDNA breaks

high low undetectable

Pairing of Cas9 SmartNickase

Highly precise and efficientgenome 

Double‐strandedDNA breaks

high high low

engineering by NHEJ or HDR

C 9 ll l GCas9 NullNuclease Gene regulation

no DNA breaks

‐ ‐ ‐

PrecisionX TM Cas9 All‐in‐one vector systemPrecisionX Cas9 All in one vector system

Key FeaturesKey Features• All‐in‐one vector system including Cas9 protein and gRNA. This 

maximizes the chances of successful cellular delivery of all necessary CRISPR/Cas components

• Pre‐linearized vector is ready‐to‐use, no need to prepare or modify the vector backbone

• Rapid, highly‐efficient cloning with low background (~99% cloning efficiency)

• Cloning compatibility the same gRNA insert can be easily exchanged• Cloning compatibility – the same gRNA insert can be easily exchanged into other Cas9 all‐in‐one vectors

• H1 promoter is used to drive gRNA expression, which allows target any genomic loci in the form of N20NGG with no constrains on transcription initiating site.

• H1 promoter displayed less off‐target effect in previous RNAip p y g papplications compared to U6 promoter

Available Cas9 All‐in‐one vectorsAvailable Cas9 All in one vectorsCat# Description Size

CAS900A-1 EF1-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn

CAS920A-1 CAG-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn

CAS940A-1 CMV-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn

CAS960A-1 MSCV-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn

CAS980A-1 PGK-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn

CAS800A-1 EF1-hspCas9 (D10A) -H1-gRNA linearized SmartNickase vector 10 rxn

CAS820A-1 CAG- hspCas9 (D10A) -H1-gRNA linearized SmartNickase vector 10 rxn

CAS840A-1 CMV- hspCas9 (D10A) -H1-gRNA linearized SmartNickase vector 10 rxn

CAS805A-1 EF1-hspCas9 DM-H1-gRNA linearized NullNuclease vector 10 rxn

List of ComponentsList of Components

Reagent Amount

Linearized Cas9 all-in-one Vector 10 µl

5x ligation buffer 10 µl

Fast ligase 2.5 µlFast ligase 2.5 µl

Fwd sequencing primer (5 µM): 5’ GTCATCAACCCGCTCCAAGG 3'

20 µl

How to build your own Cas9 All‐in‐one vector?

Selection of Target DNA SequenceSelection of Target DNA Sequence

5’ NNNNNNNNNNNNNNNNNNNNNGG 3’5 NNNNNNNNNNNNNNNNNNNNNGG 3

Using Cas9 Nickase to generate DSBs toUsing Cas9 Nickase to generate DSBs to increase targeting specificity

CGTAAGCTTACGCGATGCACNGG5’ 3’

ATCGGCATTGCTTACCGTTA

TAGCCGTAACGAATGGCAAT

CCN

gRNA 1

Cas9 (D10A) Nickase

‐5’

3’ Targeting site

GCATTCGAATGCGCTACGTG

CGTAAGCTTACGCGATGCAC

NCC53’

35’TAGCCGTAACGAATGGCAAT

GGN

gRNA 2

Cas9 (D10A) Nickase( )

5’‐

3’

5’3’

3’5’

5’ overhang

Choose your gRNA1 from the anti‐sense strand upstream of your targeting siteChoose your gRNA2 from the sense strand downstream of your targeting site

Design of Guide RNA Oligonucleotides

5’ TGTATGAGACCACNNNNNNNNNNNNNNNNNNNN 3’3’ ACTCTGGTGNNNNNNNNNNNNNNNNNNNNCAAA 5’

Anneal the two single‐strand DNA oligonucleotides

Materials Amount

10uM Top strand oligo 5 µl

10uM Bottom strand oligo 5 µl

Total volume 10 µl

Incubate reaction mixture at 95°C for 5 minutes, then cool down

Ligation of Oligo Duplex into VectorLigation of Oligo Duplex into Vector 

Materials Amounts

Linear vector 1 µl

Annealed oligo mix 3 µlAnnealed oligo mix 3 µl

5x ligation buffer 1 µl

Fast ligase 0 25 µlFast ligase 0.25 µl

Total volume 5.25 µl

Mix reaction well and incubate for 5-7Mix reaction well and incubate for 5 7minutes at room temperature

If you are making several constructs at the same time, we strongly recommend adding ligase and buffer separately and individually to the linearized vector

TransformationTransformation

• Add a vial of competent cells to the ligation mixdd a a o co pe e ce s o e ga o

• Place cells on ice for 15 minutes

• Heatshock cells at 42°C for 50 seconds, then immediately , ytransfer cells to ice for 2 minutes

• Add 250 µl SOC medium and incubate at 37°C for 1 hour with shaking

• Spread 100 µl of cultured cells on a pre‐warmed LB plate containing 50 µg/ml Kanamycin and incubate overnightcontaining 50 µg/ml Kanamycin and incubate overnight at 37°C

Transformation Results- insert +insert - insert +insert

Transformation Results

PGK-hspCas9-H1-gRNA vectorEF1-hspCas9-H1-gRNA vector

- insert +insert - insert +insert

MSCV-hspCas9-H1-gRNA vector

insert +insert

CAG-hspCas9-H1-gRNA vector

- insert +insert

CMV-hspCas9-H1-gRNA vector

Confirmation of Positive ClonesConfirmation of Positive Clones

• Pick 1 to 2 colonies, grow in LB/Kanamycin medium c o co o es, g o / a a yc ed uovernight at 37°C with shaking

• Next day, miniprep plasmid DNAs and send for sequencing using the provided sequencing primer 

(Note: Primer provided is ready to use, concentrated at 5 M i l 1 l ti )5 µM, simply use 1 µl per reaction)

• Align your raw sequencing data with Top strand primer sequencesequence

Your All-in-one vector is ready for testing!Your All in one vector is ready for testing!

PrecisionX TM Cas9 RNA systemPrecisionX Cas9 RNA system

Precision X TM Cas9 RNA systemPrecision X  Cas9 RNA systemCat# Description Size

CAS500A-1 Transfection-ready hspCas9 SmartNuclease mRNA (Eukaryotic Version) 20 µg

CAS502A-1 Transfection-ready spCas9 SmartNuclease mRNA (Prokaryotic Version) 20 µg

CAS504A-1 Transfection-ready hspCas9 (D10A) SmartNickase mRNA (Eukaryotic Nickase) 20 µg

CAS506A-1 Transfection-ready hspCas9 DM NullNuclease mRNA (Eukaryotic NullNulease) 20 µg( y )

CAS510A-1 SmartNucleaseTM Linearized T7 gRNA vector 10 rxn

CAS510A-KIT SmartNucleaseTM T7 gRNA Synthesis kit (includes CAS510A-1 and T7 IVT synthesis reagents) 1 kity g )

CAS520A-1 Transfection-ready Cas9 SmartNuclease AAVS1 gRNA 10 µg

Cas9 mRNACas9 mRNA

• a novel cap analog called Anti‐ 9

kb M hspCas9 mRNAp g

Reverse Cap Analog (ARCA) was used for Cas9 mRNA synthesis, which allows for 100% 4

56

which allows for 100% functional of Cas9 mRNA.

• No myc‐tag  2.5

3

• Two NLS

• Additional 5’UTR, 3’UTR and l A il h C 9 RNA

1..5

2

polyA tail, enhance Cas9 mRNA stability and translation efficiency 0.5

1

Linearized T7 gRNA vector( )(Cat#: CAS510A‐1)

Reagent Amount

Linearized T7 gRNA Vector 10 µl

5X Ligation Buffer 10 µl

Fast Ligase 2.5 µl

Sequencing Primer (5 µM)

5’ GCGGGCCTCTTCGCTATTAC 3'20 µl

5’ GCGGGCCTCTTCGCTATTAC 3'

T7 gRNA Synthesis Kit( )(Cat #: CAS510‐KIT)

Reagent AmountReagent AmountSmartNucleaseTM Linearized T7 gRNAVector 10 µl

5X Ligation Buffer 10 µl5X Ligation Buffer 10 µl

Fast Ligase 2.5 µlSequencing Primer (5 µM)

20 µl5’ GCGGGCCTCTTCGCTATTAC 3'

0 µ

2X NTP Buffer Mix 100 µl

T7 RNA Polymerase Mix 20 µly µ

T7 gRNA PCR primer mix (5 µM) 50 µl

DNase I (2U/µl) 10 µl

How to make your own gRNA?How to make your own gRNA?5’ NNNNNNNNNNNNNNNNNNNNNGG  3’

PAM20bp target sequenceGeneral form of target sequence

1. Design of two oligos coding target sequence

3’ NNNNNNNNNNNNNNNNNNNNCAAA  5’5’ AGGGNNNNNNNNNNNNNNNNNNNN  3’

3’ NNNNNNNNNNNNNNNNNNNNNNNCAAA  5’5’ AGGGNNNNNNNNNNNNNNNNNNNN 3’

2. Anneal of two oligos

T7 promoter Chimeric gRNA scaffold 

5’‐‐‐TAATACGACTCACTATAGGG3’‐‐‐ATTATGCTGAGTGATATCCC

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT‐‐‐3’ATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGGCAATAGTTGAACTTTTTCACCGTGGCTCAGCCACGAAAAAAAA‐‐‐5’

3. Ligation

T7 gRNA vector

4. Transformation

T7 promoter Custom gRNA5. Sequencing

5’ ‐‐‐ TAATACGACTCACTATAGGGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT‐‐‐3 ’3 ’ ‐‐‐ ATTATGCTGAGTGATATCCCNNNNNNNNNNNNNNNNNNNNCAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGGCAATAGTTGAACTTTTTCACCGTGGCTCAGCCACGAAAAAAAA‐‐‐5 ’

6. In vitro synthesis of gRNA

7. Purification of gRNA transcripts

Oligo design for T7gRNA vectorOligo design for T7gRNA vector

T t

5’ NNNNNNNNNNNNNNNNNNNNNGG 3’Target sequence

5’ AGGGNNNNNNNNNNNNNNNNNNNN 3’Oligo duplex

5 AGGGNNNNNNNNNNNNNNNNNNNN 33’ NNNNNNNNNNNNNNNNNNNNCAAA 5’

Using Cas9 Nickase to generate DSBs toUsing Cas9 Nickase to generate DSBs to increase targeting specificity

CGTAAGCTTACGCGATGCACNGG5’ 3’

ATCGGCATTGCTTACCGTTA

TAGCCGTAACGAATGGCAAT

CCN

gRNA 1

Cas9 (D10A) Nickase

‐5’

3’ Targeting site

GCATTCGAATGCGCTACGTG

CGTAAGCTTACGCGATGCAC

NCC53’

35’TAGCCGTAACGAATGGCAAT

GGN

gRNA 2

Cas9 (D10A) Nickase( )

5’‐

3’

5’3’

3’5’

5’ overhang

Choose your gRNA1 from the anti‐sense strand upstream of your targeting siteChoose your gRNA2 from the sense strand downstream of your targeting site

gRNA SynthesisgRNA Synthesis

gRNA Template PreparationgRNA Template Preparation1. PCR

2 Li i ti f RNA t t2. Linearization of gRNA construct

PCR of gRNA templatePCR of gRNA templateReagent Amount

5 HF B ff 10 l5×HF Buffer 10 μldNTP Mix 1 μlT7 gRNA PCR primer mix (5μM) 5 μlPl id T l t ( iti T7Plasmid Template (positive T7 gRNA construct)

10-50 ng

Phusion DNA Polymerase 0.3 μlNuclease Free H O to 50 μl

Cycle(s) Temperature Time1 98 °C 3 min

Nuclease-Free H2O to 50 μl

3098 °C 30 s55 °C 30 s72 °C 15 s

1 72 °C 10 min4 °C hold

Linearization of gRNA constructLinearization of gRNA construct

The positive gRNA construct can be linearizedwith EcoRI to be used as the template for in vitro gRNA synthesisvitro gRNA synthesis.

Circular plasmid templates will generate extremely long, heterogeneous RNA transcripts. We highly recommend to examine the linearized template DNA on a gel tothe linearized template DNA on a gel to confirm that cleavage is complete.

gRNA in vitro synthesisgRNA in vitro synthesis

Reagent AmountReagent AmountNuclease-free water x μl2x NTP Buffer Mix 10 μlPurified Linearized Template DNA x μl *T7 RNA Polymerase Mix 2μlTotal reaction volume 20 μlμ*Use 0.3-0.5 μg PCR-product template or ~1 μg linearized plasmid template.

Incubate at 37 °C for 4-6 hours

Treat with DNase I at 37 °C for 10 min,eat t ase at 3 C o 0 ,then heat inactivate at 75 °C for 10 min

Purification of gRNA transcriptsPurification of gRNA transcripts

1) Spin Column Purification1) Spin Column Purification

2) h l hl f i d h l2) Phenol‐chloroform Extraction and Ethanol Precipitation

• We do not recommend using LiCl for gRNAg gtranscript precipitation.

Analysis of guide RNA transcriptsAnalysis of guide RNA transcripts

• The size of the gRNA transcripts can beThe size of the gRNA transcripts can be analyzed by running an aliquot of the reaction on formaldehyde‐based denaturing agaroseon formaldehyde based denaturing agarosegel.

• The concentration of the gRNA transcripts can• The concentration of the gRNA transcripts can be determined by reading the A260 of a diluted aliquotaliquot. 

Your gRNA is ready for testing!

Genome editing by PrecisionXTMGenome editing by PrecisionXCas9 Systemy

G l tGene replacement

Gene correction/repair

Gene regulation

Gene replacement using Cas9 SmartNuclease construct

Cas9 SmartNuclease targeting Luciferase gene

Gene replacement using Cas9 SmartNuclease construct

Gene replacement using Cas9 SmartNuclease construct

Luciferase Assaytiv

ity 120.00%

fera

se a

ct

60.00%

80.00%

100.00%

lativ

e Lu

ci

20.00%

40.00%

Rel 0.00%

GFP+RFP‐ GFP+RFP+

Gene RepairGene Repair

CAS605 positive control kit

• EF1 hCas9 H1 AAVS1 gRNA (CAS601A 1)• EF1‐hCas9‐H1‐AAVS1 gRNA (CAS601A‐1)

• EGIP 293T reporter cell line

• AAVS1/GFP rescue donor

• primers for Surveyor assayp y y

Gene repair using Cas9 SmartNuclease construct

Gene repair using Cas9 SmartNuclease construct

All‐in‐ one Cas9 (D10A) SmartNickaseAll in one Cas9 (D10A) SmartNickaseand Cas9 DM NullNuclease

1 2 3 4 5

1. DNA marker2. EF1-hspCas9-H1-AAVS-gRNA3. EF1-hspCas9 (D10A)-H1-AAVS-gRNA4. EF1-hspCas9-DM-H1-AAVS-gRNA5. Negative control EGIP cellg

All‐in‐ one Cas9 (D10A) SmartNickased C 9 DM N llN land Cas9 DM NullNuclease

EF1-hspCas9-H1-AAVS gRNA + Donor

EF1-hspCas9 (D10A)-H1-AAVS gRNA + Donor

EF1-hspCas9- DM-H1-AAVS gRNA + Donor

Paired EF1-hspCas9 (D10A)-H1-AAVS gRNAs + Donor

Gene repair using PrecisionX TM

Cas9 RNA system

Gene repair using PrecisionX TM

C 9 RNACas9 RNA system

hspCas9 mRNA + AAVS gRNA+ Donor+ Donor

hspCas9 (D10A) mRNA + paired AAVS gRNAs+ Donor

Gene regulation using Cas9 DM N llN lNullNuclease

hspCas9 DM NullNuclease

ase

activ

ity

0 8

1

1.2

1.4

hspCas9 DM NullNuclease

Rel

ativ

e lu

cife

ra

0

0.2

0.4

0.6

0.8

R

DM CMV‐T CMV‐B GFP‐T GFP‐B Luc‐T Luc‐B

Q&AQ&A

• Q: How many guide RNA constructs do you have toQ: How many guide RNA constructs do you have to design to target a DNA sequence of interest?

• A: Due to the unpredictable efficacy of a particular guide RNA construct, for optimal results we suggest g p ggdesigning multiple (2 or more) constructs targeting a particular DNA sequence of interest

Q&AQ&A

• Q: We designed a guide RNA construct to transfect into target cells and there is no evidence of activity. What are the possible reasons for this?

• A: There are many possibilities of why a particular guide RNA does not show any measureable effects.  Some of the possibilities include the following: 

1) Poor transfection efficiency of target cells. 

2) Errors in guide RNA design. 

3) Errors in Surveyor assay

) ( ) d4) Mutation(s) in DNA sequence targeted

Q&AQ&A

• I want to use paired nickases for genome editing, what’ a o use pa ed c ases o ge o e ed g, are the parameters that I should be concerned.

• When use paired nickases for genome editing, please pay attention to the following parameters

1) the gRNA pairs must create 5’ overhangs 

2) make sure each gRNA is able to efficiently induce indelswhen coupled with wide‐type Cas9. 

3) The distance between gRNA pairs should range from3) The distance between gRNA pairs should range from ‐8bp to 100bp

Q&AQ&A

• Q: Why use HR for genome engineering application?Q y use o ge o e e g ee g app ca o

• A: There are couple advantages to use HR for genome engineeringengineering

1) Precise and controllable1) Precise and controllable

2) Any desired change can be implemented 

3) Well‐established enrichment/selection strategy3) Well‐established enrichment/selection strategy

Q&AQ&A

• Q: We want to perform HR to modify our gene using the Q e a o pe o o od y ou ge e us g eCas9 SmartNuclease system, but we do not have the corresponding donor vectors.  What are our options in hi ?this case?

A Th l ti f f i HR i• A: There are several options for performing HR using a donor vector accompany with  Cas9 SmartNuclease system in cells.y

Options for performing HROptions for performing HR• Option #1 – Design an HR donor vector containing the DNA 

f t t b i t d difi d i t t t ll f kifragment to be inserted or modified into target cells, franking with 5’ and 3’ arms homologous (~800bp) to the desired target region, and may contain selection or fluorescent markers for selection of cells after HR.

• Option #2 – SBI will provide a full suite of off‐the‐shelf HR cloning vectors containing multiple MCS for cloning of homologycloning vectors containing multiple MCS for cloning of homology arms and insert sequences, as well as selectable fluorescent and antibiotic selection markers.  Please inquire for availability of these vectors. 

• Option #3 – SBI can build a custom HR donor vector targeting any sequence of interest as part of our custom cloning serviceany sequence of interest as part of our custom cloning service

PrecisionX™ HR Targeting VectorsPrecisionX  HR Targeting Vectors

Gene knock-out/correction

Gene knock-in

PrecisionX™ HR Targeting VectorsPrecisionX  HR Targeting Vectors

Gene tagging

Q&AQ&A

• Q: How’s CRISPR‐Cas9 system in comparisonQ: How s CRISPR Cas9 system in comparison of TALENs and ZFNs?

• A: Generally speaking, CRISPR‐Cas system id l d ZFN dprovides several advantages over ZFNs and 

TALENs

CRISPR‐Cas vs. TALENs & ZFNsCRISPR Cas vs. TALENs & ZFNsAdvantages:

Easier to implement

Ability to nick either or both  DNA strand

fMore frequent target sites 

higher targeting efficiency

Possibly higher off target effect

Disadvantages:

Can be alleviated by paired Cas9 nickases

Nature Biotechnology 2012; 30(9): 836-838

LicensingLicensing

• SBI has pending patent applications related toSBI has pending patent applications related to the PrecisionX TM Cas9 Products.  SBI does not require a use‐license for research purposes forrequire a use license for research purposes for non‐profit or for‐profit institutions. For commercial applications please contact SBIcommercial applications, please contact SBI for more information

In summaryIn summary

• CRISPR‐Cas9 system defines a new class of genome S as9 sys e de es a e c ass o ge o eengineering tools

• SBI’s  PrecisionX TM Cas9 system allows genome targeting and modification in a simple, flexible, scalable and efficient way with desired high specificity. 

SBI id it f d t t h• SBI provides a suite of products as your one stop shop for genome engineering

System Biosciences (SBI)System Biosciences (SBI)www.systembio.com

System Biosciences (SBI)265 North Whisman Rd.Mountain View, CA 94043T l 650 968 2200Tel: 650-968-2200Fax: 650-968-2277E-mail:General Information: info@systembio comGeneral Information: info@systembio.comTechnical Support: tech@systembio.comOrdering information: orders@systembio.com