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wellgenetics.com [email protected] +8862-2651-1809 +8862-2782-9911 CRISPR Genome Editing CRISPR Genome Editing Embryo Microinjection Embryo Microinjection Service Catalog Service Catalog CRISPR Genome Editing Embryo Microinjection Service Catalog CRISPR Genome Editing Services (Drosophila) Embryo Microinjection Services (Drosophila & Mosquito)

CRISPR Genome Editing Embryo Microinjection … Genome Editing Embryo Microinjection Service Catalog ... research institutes. ... CRISPR Strategy Proposal, Target Site Design,

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Page 1: CRISPR Genome Editing Embryo Microinjection … Genome Editing Embryo Microinjection Service Catalog ... research institutes. ... CRISPR Strategy Proposal, Target Site Design,

wellgenetics.com [email protected]+8862-2651-1809 +8862-2782-9911

CRISPR Genome EditingCRISPR Genome EditingEmbryo MicroinjectionEmbryo MicroinjectionService CatalogService Catalog

CRISPR Genome EditingEmbryo MicroinjectionService Catalog

CRISPR Genome Editing Services(Drosophila)

Embryo Microinjection Services(Drosophila & Mosquito)

Page 2: CRISPR Genome Editing Embryo Microinjection … Genome Editing Embryo Microinjection Service Catalog ... research institutes. ... CRISPR Strategy Proposal, Target Site Design,

Free Consulting Service: Strategy & Plan

Below are 4 most popular genome editing requests: Gene Disruption, Reporter Knock-in, Tag Knock-in,

and Seamless Editing. Any special editing request other than listed is welcomed. WellGenetics scientists

will suggest strategies to meet specically to your needs and propose a most practical and economic way

to achieve what you want!

A

dsDNA Donor

// //

loxP loxP

stop Selection Marker

HDR

Genetic Screening

// //stop Selection Marker

B

Cassetteoptions for Ⓐ

B1loxP

stop

loxP

stopattPX

B2

B3

loxP loxP

attP

loxP loxP

attP attP

B4

B5

Reporter

GFP, Gal4,Gal4::VP16,LexA ...

loxP loxP

attP

Reporter

GFP, Gal4,Gal4::VP16,LexA ...

loxP loxP

attP attP

Cassetteoptions for Ⓑ

Gene Disruption

// //

HDR

Genetic Screening

dsDNA Donor

attP attP

// //

loxP loxP

WellGenetics is dedicated to providing researchers professional

services in generating transgenes and gene knockout/knockin in

y models. We are an independent company from any academic

research institutes. Each project will be designed, executed, and

supervised by postdoctoral scientists, thus to ensure the quality

and reliability of entire experiment process.

Simply let us know the name of gene and how you want it to be

edited. We will propose the best strategy and do the rest of steps

for you - from Design to Fly! Please see service lists below.

Fly Genome Editing Expert — WellGenetics

A2loxP

stop

loxP

stopattPX

A3 hsp70 wReporter

GFP, Gal4,Gal4::VP16,LexA ...

loxP loxP

BglII

A1loxP

stop

loxP

stop

T2A

Selection Marker

Selection Marker attPattPhsp70 w

hsp70 w

3xP3 RFP

3xP3 RFP

3xP3 RFP

3xP3 RFP

3xP3 RFP

Selection Marker Options: SM1 3xP3 RFP3xP3-RFP SM2 hsp70 whsp70-w cDNA

Page 3: CRISPR Genome Editing Embryo Microinjection … Genome Editing Embryo Microinjection Service Catalog ... research institutes. ... CRISPR Strategy Proposal, Target Site Design,

Reporter Knock-in

E Gene Manipulations: T-GEM/ CRIMIC (CRISPR + MiMIC) (Diao et al., 2015 Cell Report)

// //coding intron

T-GEM Plug In

attP attPSASA SASDattP

Use Crispr/Cas with T-GEM

attP attPattP

pASAattP attPpASAattP

Use "off-the-shelf" MiMIC line OR

// //attP attPattP

attP attBattB

QF2attP attBattB

LexA::QFAD

attP attBattB

3XGal80

attP attBattB

Gal4DBD

attP attBattB

p65AD

Trojanexon

Cassetteoptions for Ⓔ E1 3xP3Gal4 pASA RFP

loxP loxP

attP attP

D Cassetteoptions for Ⓓ

D1 Reporter

GFP, Gal4,Gal4::VP16,LexA ...

loxP loxP

BglII

C C1

C2

C3

Reporter

GFP, Gal4,Gal4::VP16,LexA ...

loxP loxP

Reporter

loxP loxP

attP

Reporter

loxP

attP attP

Cassetteoptions for Ⓒ

T2A

T2A Gal4

// //

dsDNA Donor

loxP loxP

Reporter

// Reporter //Selection Marker

Selection Marker

HDR

Genetic Screening

// //

// //

dsDNA Donor

loxP loxP

Reporter

Reporter

T2A Selection Marker

Selection MarkerT2A

HDR

Genetic Screening

T2A

loxP

Tag Knock-in

GF

// //

Cre/loxP Excision (Add-on Service)

//

//

//

dsDNA Donor

loxP loxP

Tag

Tag

//Tag

HDR

Genetic Screening

Selection Marker

Selection Marker

HA, APEX2, GFP ...

// //

Cre/loxP Excision (Add-on Service)

//

//

//

dsDNA Donor

loxP loxP

Tag

//

HDR

Genetic Screening

Selection Marker

HA, APEX2, GFP ...

Tag

Tag

hsp70 w

hsp70 w

hsp70 w

hsp70 wN-terminal Reporter

C-terminal Reporter

N-terminal Tagging N-terminal Tagging

Linker peptide after excision

Selection Marker

Page 4: CRISPR Genome Editing Embryo Microinjection … Genome Editing Embryo Microinjection Service Catalog ... research institutes. ... CRISPR Strategy Proposal, Target Site Design,

Tag Knock-in (continue)

J

I

L

//

// //

//

ssODN or dsDNA Donor

Seamless Editing (Point Mutation, Fragment Knock-in/ Knock-out/ Replacement)

HDR

PCR/ HRMA/ Digestion Screening (Add-on Service)

M

//

//

//

PBac Transposition (Add-on Service)

dsDNA Donor

PiggyBac-based Scarless System(Developed by O'Connor-Giles' Lab)

TTAA

3xP3 DsRed TTAA

//

TTAA //

//

TTAA

3xP3 DsRed TTAATTAA

HDR

Genetic Screening

K

// //

dsDNA Donor

Tag

//

HA or Flag

loxP loxP

Tag //

HDR

Genetic Screening

Selection Marker

Selection Marker

// //

Cre/loxP Excision (Add-on Service)

dsDNA Donor

// //Tag SDSA

// //Tag SDSA

loxP loxP

Tag SDSA

HDR

Genetic Screening

Selection Marker

Selection Marker

HA, APEX2, GFP ...

gRNA Transgenic Fly

Generation Service

Microinject gRNA plasmid into

requested landing site and set

up the gRNA transgenic stock.

Gene Synthesis Service

To avoid confusion of polymor-

phism or PCR errors occurred in

PCR cloning, you may choose to

synthesize homology arms.

Selection Marker Excision Service

To excise selection marker oxed by

loxP/PBac, we provide cre/loxP

excision and PBac transposition

services.

Customized Cassette

Cloning Service

Design and construct

cassette for your specic

research needs.

PCR Screening Service

Individual F1 progeny is back-

crossed to balancer. We then

perform single-y genomic

PCR on each fertile F1 ies.

HRMA Screening

Individual F1 progeny is back-crossed to

balancer for high resolution melting analysis

(HRMA). Using HRMA to screen for single-

nucleotide mismatches or indel mutations.

Digestion Screening

Individual F1 progeny is back-crossed to balancer.

We then perform single-y genomic PCR on each

fertile F1 ies. PCR products will be puried and cut

by endonuclease and analyzed by gel eletrophoresis.

Add-on Services for CRISPR Genome Editing

H Internal Tagging C-terminal Tagging

// //

Cre/loxP Excision (Add-on Service)

dsDNA Donor

// //

loxP

Tag

loxP

// //

HDR

Genetic Screening

Selection Marker

HA, APEX2, GFP ...

Tag Selection Marker

Tag

// //

Cre/loxP Excision (Add-on Service)

dsDNA Donor

// //Tag

loxP

Tag

loxP

// //Tag

HDR

Genetic Screening

Selection Marker

Selection Marker

HA, APEX2, GFP ...

C-terminal Tagging C-terminal Tagging

Linker sequence after excision Linker peptide after excision

Page 5: CRISPR Genome Editing Embryo Microinjection … Genome Editing Embryo Microinjection Service Catalog ... research institutes. ... CRISPR Strategy Proposal, Target Site Design,

CRISPR Service Packagewith Selection Marker -or- with PCR Screening

WellGenetics provides CRISPR Service Package including Design, Cloning,

Microinjection, Screening and Stock Establishment to bring you gene-edited

ies. Simply let us know the name of gene and how you want it to be edited. We

propose the best strategy and do the rest of steps for you - from Design to Fly.

Using homology-dependent repair (CRISPR/HDR), we can knockin desired

cassette in combination with cuts by 1 or 2 guide RNAs. We welcome any project

that requires customized designs to t your specic research needs. Feel free to

show us your interests in CRISPR y!

Workow of CRISPR Service Package

CRISPR Service Package with Selection Marker CRISPR Service Package with PCR Screening

Step 2

Step 1

Select guide RNA target sites at your preferred region(s)

Propose and quote you the best suggestions to your needs

Step 3

Step 4

Design repair template as donor carrying chosen knock-in cassette

Conrm target sites by genomic PCR in the injection strain

Step 5 Guide RNA plasmid construction and plasmid donor construction or ssODN donor synthesis

Step 6 Plasmids DNA preparation & purication

Step 7 200-embryos microinjection

Step 8 G0 cross

Step 9 F1 Screening by visible marker(NO additional charge)

F1 Screening by PCR(with additional charge)

Step 10 Genomic PCR validation

Step 11 Positive line isogenization and balancing

Step 12 If the rst trial fails, free second round microinjection with optimization

If both trials fail, we only charge half of the package price* *Terms and Conditions apply

Package

CRISPR Service PackageYou Provide: Name of Gene & Editing Ideas. We Do the Rest: CRISPR Strategy Proposal, Target Site Design,

Target Site Conrmation, Cloning, Microinjection, Screening , Molecular Validation, and Balancing.

1 week 2+4 weeks 2 weeks 4-5 weeks 4-5 weeks

Step 2-3. Design Step 4-5. Cloning Step 6-7. Microinjection Step 8-9. Screening Step 10. Validation Step 11. Balancing

Our Clients... It is our great honor to make contributions to your research projects

sni t i t u a l r o s et

B i o l o g i e

National Chung Hsing University

Guarantee

// //

Page 6: CRISPR Genome Editing Embryo Microinjection … Genome Editing Embryo Microinjection Service Catalog ... research institutes. ... CRISPR Strategy Proposal, Target Site Design,

Drosophila & Mosquito Embryo Microinjection ServicesWellGenetics provides Fly Microinjection Services for CRISPR, PhiC31, P-element and

PiggyBac transgenesis in Drosophila melanogaster, and Mosquito Microinjection Services

for CRISPR and Transposon transgenesis in Aedes aegypti.

Embryo Microinjection

Microinjection Service Lists

PhiC31 Microinjection & Screening Services*

200-embryos microinjection, G0 cross & F1 screen (w+).

We return unbalanced positive lines.

We have VK33, VK37, attP2, attP40, ZH51C &

ZH86Fb as regular panel of landing sites. You can

also choose any other sites listed in Bloomington Stock

Center and allow two generations of amplication time.

We guarantee 1-3 transformants for plasmid at standard

size (<13kb).

Bac (P[acman]) Microinjection & Screening

Services*

600-embryos microinjection, G0 cross & F1 screen (w+).

We return unbalanced positive lines.

CRISPR Microinjection

200-embryos microinjection. We return survived larvae

after microinjection.

Mosquito Microinjection

500-embryos microinjection. We return injected embryos

after microinjection.

Non-melanogaster Microinjection

200-embryos microinjection. We return survived pupae

after microinjection.

P-element Microinjection & Screening Services*

200-embryos microinjection, G0 cross & F1 screen (w+).

We return unbalanced positive lines.

We guarantee 5-8 transformants for plasmid at standard

size (<13kb).

*NO guarantee for large plasmid (>13kb).

Add-on Services

Balancing Service

Isogenize & balance positive lines from screening.

We establish y stocks for you.

Plasmid Construction Service (<3kb)

Maximum 3 PCR-fragments and together length below 3kb.

We use High-Fidelity Phusion DNA Polymerase to amplify

the fragments and sequence through all parts we cloned in.

We return bacteria streaking plate and 4µg plasmid DNA.

BAC DNA Prep.

Qiagen Large Construct Kit for BAC DNA preperation

(>20kb) to provide high quality DNA for microinjection.

Validation service by restriction enzyme digestion or PCR

is included!

Plasmid DNA Prep.

Plasmid preperation to provide high quality DNA for

microinjection. Validation service by restriction enzyme

digestion or PCR is included!

Want to Know More about Our Services?Contact WellGenetics [email protected]+8862-2651-1809