CRISPR Genome Editing Embryo Microinjection Service Catalog

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1 CRISPR Genome Editing Embryo Microinjection Service Catalog CRISPR Genome Editing Services (Drosophila) Embryo Microinjection Services (Drosophila & Mosquito) ellgenetics.com

2 Fly Genome Editing Expert WellGenetics 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 ill be designed, executed, and supervised by postdoctoral scientists, thus to ensure the quality and reliability of entire experiment process. Simply let us kno the name of gene and ho you ant it to be edited. We ill propose the best strategy and do the rest of steps for you - from Design to Fly! Please see service lists belo. Free Consulting Service: Strategy & Plan Belo are 4 most popular genome editing requests: Gene Disruption, Knock-in, Knock-in, and Seamless Editing. Any special editing request other than listed is elcomed. WellGenetics scientists ill suggest strategies to meet speci cally to your needs and propose a most practical and economic ay to achieve hat you ant! Gene Disruption A options for A A1 A2 X BglII A3 T2A GFP, Gal4, B options for B B1 X B2 B3 B4 GFP, Gal4, B5 GFP, Gal4, Options: SM1 - SM2 - cdna

3 Knock-in C N-terminal options for C C1 GFP, Gal4, C2 C3 D C-terminal T2A BglII options for D D1 T2A GFP, Gal4, T2A E Gene Manipulations: T-GEM/ CRIMIC (CRISPR + MiMIC) (Diao et al., 2015 Cell Report) Use "off-the-shelf" MiMIC line OR Use Crispr/Cas ith T-GEM options for E E1 SA T2A Gal4 pa SA SA SD SA T2A Gal4 pa coding intron T-GEM Plug In LexA::QFAD QF2 Gal4DBD Trojan exon 3XGal80 p65ad Knock-in F N-terminal ging G N-terminal ging HA, HA, Cre/ Excision (Add-on Service) Cre/ Excision (Add-on Service) Linker peptide after excision

4 Knock-in (continue) H Internal ging I C-terminal ging SA SD HA, HA or Flag SA SD Cre/ Excision (Add-on Service) SA SD J C-terminal ging K C-terminal ging HA, Linker sequence after excision Cre/ Excision (Add-on Service) HA, Linker peptide after excision Cre/ Excision (Add-on Service) Seamless Editing (Point Mutation, Fragment Knock-in/ Knock-out/ Replacement) L M PiggyBac-based Scarless System (Developed by O'Connor-Giles' Lab) ssodn or PCR/ HRMA/ Digestion Screening (Add-on Service) DsRed DsRed PBac Transposition (Add-on Service) Add-on Services for CRISPR Genome Editing Customized Cloning Service Design and construct cassette for your speci c research needs. 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 polymorphism or PCR errors occurred in PCR cloning, you may choose to synthesize homology arms. Excision Service To excise selection marker oxed by /PBac, e provide cre/ excision and PBac transposition services. PCR Screening Service Individual F1 progeny is backcrossed 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 singlenucleotide 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 ill be puri ed and cut by endonuclease and analyzed by gel eletrophoresis.

5 CRISPR Service Package ith -or- ith PCR Screening WellGenetics provides CRISPR Service Package including Design, Cloning, Microinjection, Screening and Stock Establishment to bring you gene-edited ies. Simply let us kno the name of gene and ho you ant 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/), e can knockin desired cassette in combination ith cuts by 1 or 2 guide RNAs. We elcome any project that requires customized designs to t your speci c research needs. Feel free to sho us your interests in CRISPR y! Work o of CRISPR Service Package Package CRISPR Service Package ith CRISPR Service Package ith PCR Screening Step 1 Propose and quote you the best suggestions to your needs Step 2 Select guide RNA target sites at your preferred region(s) Step 3 Design repair template as donor carrying chosen knock-in cassette Step 4 Con rm 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 & puri cation Step embryos microinjection Step 8 G0 cross Step 9 F1 Screening by visible marker F1 Screening by PCR (NO additional charge) (ith 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 ith optimization Guarantee If both trials fail, e only charge half of the package price* *Terms and Conditions apply CRISPR Service Package You Provide: Name of Gene & Editing Ideas. We Do the Rest: CRISPR Strategy Proposal, Target Site Design, Target Site Con rmation, Cloning, Microinjection, Screening, Molecular Validation, and Balancing. Step 2-3. Design Step 4-5. Cloning 1 eek Step 6-7. Microinjection 2+4 eeks 2 eeks Step 8-9. Screening 4-5 eeks Step 10. Validation 4-5 eeks Our Clients... It is our great honor to make contributions to your research projects alr o se institut B i o l o g i e National Chung Hsing University Step 11. Balancing

6 Embryo Microinjection Drosophila & Mosquito Embryo Microinjection Services WellGenetics 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. Microinjection Service Lists PhiC31 Microinjection & Screening Services* 200-embryos microinjection, G0 cross & F1 screen (+). We return unbalanced positive lines. We have VK33, VK37, 2, 40, ZH51C & ZH86Fb as regular panel of landing sites. You can also choose any other sites listed in Bloomington Stock Center and allo to generations of ampli cation time. We guarantee 1-3 transformants for plasmid at standard size (<13kb). P-element Microinjection & Screening Services* 200-embryos microinjection, G0 cross & F1 screen (+). We return unbalanced positive lines. We guarantee 5-8 transformants for plasmid at standard size (<13kb). *NO guarantee for large plasmid (>13kb). Bac (P[acman]) Microinjection & Screening Services* 600-embryos microinjection, G0 cross & F1 screen (+). We return unbalanced positive lines. CRISPR Microinjection 200-embryos microinjection. We return survived larvae after microinjection. Non-melanogaster Microinjection 200-embryos microinjection. We return survived pupae after microinjection. Mosquito Microinjection 500-embryos microinjection. We return injected embryos after microinjection. 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 belo 3kb. We use High-Fidelity Phusion DNA Polymerase to amplify the fragments and sequence through all parts e cloned in. We return bacteria streaking plate and 4µg plasmid DNA. Plasmid DNA Prep. Plasmid preperation to provide high quality DNA for microinjection. Validation service by restriction enzyme digestion or PCR is included! 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! Want to Kno More about Our Services? Contact WellGenetics info@ellgenetics.com