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*Nature : “CRISPR mouse model boom, rat model renaissance” Nature Biotechnology 34, 893-894, September (2016) and " editing at CRISPR speed", Nature Biotechnology 32,

309–312, April (2014).

ASC’s Editing Technologies Overview

Site-Specifc! CRISPR/Cas9 Fast! Knockin! attP 3-6 months! Cas9 Guide RNA (FOPNJD%/"

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ASC has generated hundreds of models using CRISPR technology. Our scientists have optimized unique CRIPSR/Cas9 design strategies, protocols and validation methods. Take advantage of our precise genome editing technologies, fast turnaround and reasonable cost.

Step: CRISPR Validation of Breeding, F1 Germline Project Design, Targeting Pronuclear CRISPR Housing and Transmission Strategy Vector .JDSPJOKFDUJPO Elements Genotyping (if desired) & Quotation Construction

Deliverable: tFounder(s) with desired or genomic modifcation. t.JMFTUPOFSFQPSUTBOEa fnal report on the CRISPR project, including the original targeting strategy, details, and genotyping results.

Case Studies 1. Conditional Knockout Goal: To generate a conditional knockout mouse model with LoxP sequences inserted in intron 1 and downstream of 3’ UTR of the desired locus, using CRISPR-Cas9. How? This conditional knockout mouse model was generated using CRISPR Technology. The mice born from the microinjection were screened for the presence of LoxP sites at designated locations using PCR (Figure 1a). The potentially positive animals were then confrmed to have the LoxP sequences by sequencing the modifed regions in the mouse genomic locus (Figure 1b).

Figure 1a. PCR results of mice born after micro- Founder #1 Founder #2 Founder #1 Founder #2 injection of the with CRISPR cocktail. Two out of twelve mice were identifed as found- ers and showed the expected fragment shifts for both 5’ and 3’ LoxP insertions. A LoxP insertion at the 5’ site, or intron 1 produced a 513bp PCR fragment (blue box; WT: 473bp) and LoxP insertion at the 3’-targeting site produced a 539bp PCR fragment (red box; WT: 499 bp).

Sequencing of 5’ PCR fragment LoxP LoxP sequence

Founder mice

LoxP LoxP sequence

Founder mice

Figure 1b. Representative sequence analyses of founder mice confrms LoxP insertion at 5’ and 3’ locations at the desired genome locus. in vivo Models in vivo models

2. Region-Specifc Knockout Goal: To delete a 587 bp fragment from a specifc site in C57Bl/6 mouse genome using CRISPR. How? The knockout mouse model was generated using an optimized three step procedure: (1) a mixture of active guide RNA molecules (gRNAs) and qualifed Cas9 mRNA was injected into the cytoplasm of C57BL/6 embryos; (2) The new mice born from the microinjection were screened using PCR; (3) the positive animals were confrmed to have deletion by sequencing the modifed region in the desired mouse locus.

Figure 2. .JDF        XFSF JEFOUJöFE BT ' HFSNMJOF USBOTNJUUFE BOJNBMT carrying the ~ 600 bp deletion. The lower band was extracted, purifed and sequenced. The sequence results showed that the deletion removed the entire targeted exon.

3. Constitutive Knockout Goal: To generate a constitute knockout (KO) of a gene of interest in C57Bl/6 mouse genome using CRISPR. How? The knockout mouse model was generated by microinjection of a cocktail of CRISPR elements into cytoplasm of C57BL/6 mouse embryos. Ten mice born from the microinjection were screened using PCR and sequencing. Four out of the ten mice were identifed to have a constitutive KO in the gene of interest with a deletion in exons 6 to 8: mice #4,5 and 7 were identifed to have heterozygous deletion genotype while mouse #10 was a homozygous deletion mutant. M wt 1 2 3 4 5 6 7 8 9 10 wt M Figure 3a. Genotype screening for 10 mice born after microinjection of a CRISPR cocktail designed to generate a constitutive KO mouse model. (a) PCR products for NJDFBOEBXJMEUZQF XU DPOUSPM(FOF3VMFSLC%/"NBSLFSVTFE MBOF.

WT Figure 3b. Sequence Mutant alignment for mouse #10 (bottom) showing !!!!!!!! deletion in gene of interest when compared WT to wild type (top) Mutant

4. Large Fragment Knock-in Goal: To insert a 2 kb DNA fragment (gene of interest) at a specifed locus in the mouse genome using CRISPR/Cas9. How? An optimized mixture of Cas9 gRNA mRNA and donor vector was microinjected into embryos of C57BL/6 mouse background. Using a panel of genotyping primer pairs, three out of 31 pups born after microinjection (#15, 19, and 26) were identifed as founders (F0), with the gene of interest inserted at the desired locus.

Figure 4. Agarose gel electrophoresis of PCR amplicons in F0 mice (#15, 19, and 26) with site-specifc gene knock-in. The left part of the gel shows the 5’ junction fragment (2,191 bp), and the right part of the gel shows the 3’ junction fragment  CQ  in vivo Models in vivo models

Fast! Knock-in Mice TARGATT™ Fast & Site-Specifc Knock-in Mouse Service With our proprietary technology we will generate your site-specifc (Rosa26 or H11) knock-in mouse in as fast as 3 months. Reliable: Any gene of interest can be specifcally inserted at a defned, transcriptionally active locus. We guarantee expression without any interruption in internal gene expression. Step:

CRISPR PhiC31 Pronuclear TARGATTTM ValidationIntegrase of AnimalBreeding, Care, F1 Germline ProjectProject Designdesign Targeting MicroinjectionPronuclear Housing Vectorvector ExpressionCRISPR Housing and Transmission andand Strategystrategy Microinjectionin Embryos and Constructionconstruction elementsand Genotyping (if desired) Purifcation Genotyping

Deliverable: tFounders with desired mutation or genomic modifcation. t.JMFTUPOFSFQPSUTBOEa fnal report on the TARGATT™ project, including the original targeting strategy, microinjection details, and genotyping results.

Case Studies 1. Site-specifc knock-in of a coat color gene in mice Figure 1a. Left: H11;C57BL/6 mice used for donor embryos. Center: Site-specifc gene knockin founders. Right: Germline transmission in F1 mouse. Ref: Guenther et al., “A molecular basis for blond hair color in Europeans”, Nature , doi:10.1038/ng.2991.

Figure 1b. Twenty-three pups were born from one microinjection experiment. *Two positive founders were identifed by using PCR primers (PCR1, 2, 3, 4, 5). To confrm the insertion is site specifc at H11.

More sample studies are available upon request.

References: Description of the technology: Application for mice generated by TARGATT™: Zhu, F., et al. (2014). Nucleic acids research, 42(5), e34-e34. Booze, M. L., et al. (2016). Free Radical and Medicine, 99: 533-543. Tasic, B., et al. (2011). PNAS USA, 108(19), 7902–7907. Feng, D., et al. (2016). The Journal of Clinical Investigation, 126(6). Commentary, comparison with other transgenic methods: Park, K. E., et al. (2016). International journal of molecular sciences,17(6), 810. Rossant, J., et al. (2011). PNAS USA 108(19), 7659–7660. Sun, N., et al. (2015). Measuring in vivo mitophagy. Molecular cell, 60(4), 685-696 Tet inducible mice generated by TARGATT™: Guenther, CA., et al. (2014). Nature genetics, 46(7), 748-752 Fan, X., et al. (2012). Endocrinology, 153(11), 5637–5644. Devine, WP., et al. (2014). eLife, 3, e03848. Advantage of Hipp11 (H11) locus: Villamizar, C. A. (2014) UT GSBS Dissertations and These (Open Access). Paper 508 (2014) Hippenmeyer, S., et al. (2010). Neuron, 68(4), 695–709. Fogg, PC., et al. (2014). Journal of molecular biology, 426(15), 2703-2716 in vivo Models in vivo models

2. TARGATTTM Large Fragment Knock-in Goal: To insert a large (8.5kb) DNA fragment into the H11 safe harbor locus in FVB mice using TARGATT™ integrase based technology.

425N Frt-R M 1 2 3 4 5 M H11 U attP1 attP2 attP3 mCherry-F RP552N 425N PR436N Frt-R mCherry-F mCherry-R CAG-R GFP H11 U attL CAG Gene A eGFP polyA Gene B mCherry polyA attR

U Unique Sequence Primers LoxP Stop Cassette IRES Frts Genomic Sequence Figure 2b. Genotyping of gene A founder Figure 2a. Transgene integration and genotyping scheme for gene A transgenic Model. 1614#1 (a) PCRs 1 to 5 as described in Method Upper panel: genomic sequence with H11P3 landing pad; lower panel: founder with (Lanes 1 to 5); (b) 100bp ladder DNA marker gene A transgene inserted at the 1, 3-attP site used in lanes M. Fast & Site-Specifc Knock-in Mouse Do-it-yourself Products Using our novel TARGATT™ system, a gene of interest can be inserted at a well-characterized, transcriptionally active locus in the mouse genome with guaranteed transgene expression. Make your own TARGATT™ knock-in mouse by purchasing the TARGATT™ Mouse from Charles River, available in FVB and C57BL/6 backgrounds. Essential components, including TARGATT™ Plasmids and Transgenic kits, are available directly from us. Ask us for a list of core facilities currently using this system, including UCSF, NIH, Max Planck, Harvard, Columbia and more!

Step 1. Purchase Plasmids from ASC Step 3. Purchase Transgenic Kits from ASC TARGATT™ Plasmids for injection AST-3042 TARGATT™ 2 (CAG + Poly A) TARGATT™ Transgenic Kit AST-3043 TARGATT™ 3 (no promoter + MCS) AST-1003 Transgenic Kit (5 rounds of ) AST-3050 TARGATT™ 6.1 (CAG-L4SL-MCS-PolyA) AST-1004 Transgenic Kit (2 rounds of microinjections) AST-3047 TARGATT™ 7 (PGK-MCS-PolyA) AST-3048 TARGATT™ 8 (PCA-MCS-PolyA) AST-3051 TARGATT™ 9 .1 (PCA-L4SL-MCS-PolyA) Step 4. Confrm sequence with Genotyping Kit Step 2. Purchase attP mice from Charles River Site-Specifc! TARGATT™ Mouse (Charles River) Fast! Knockin! STRAIN CODE #537 (FVB), #549 (C57BL/6) AST-2005 H11 Mouse Genotyping Kit Ordering: 1.800.LABRATS AST-2006 Rosa26 Mouse Genotyping Kit

FAQ for TARGATT™ System (Selected) 1. Can I create models to over-express a gene of interest? Yes, TARGATT™ system is ideal for gene over-expression. Diferent promoters, e.g., tissue-specifc promoters or ubiquitous promoters, and inducible systems (Tet On/Of, loxP-stop-loxP) can be used for tissue-specifc, ubiquitous, or inducible gene expression.

2. Can I use TARGATT™ system to create transgenic mice with tissue-specifc gene expression? Yes, TARGATT™ system can be used to generate tissue-specifc transgenic mouse models. Just use a tissue-specifc promoter to drive- -theM transgene expression. Alternatively, a loxP-stop-loxP cassette can be placed between a ubiquitous promoter and the transgene. Upon crossing with tissue-specifc Cre mice, the transgene will be expressed in that particular tissue.

3. What promoters are used to drive gene expression? Any defned promoters provided by the customer or published in the literature can be used.

4. Can I integrate a reporter gene? What kind of reporter do you recommend? Yes, you can express any reporter genes such as GFP, DsRed, mCherry, LacZ , Luciferase, etc.

5. What is the maximum size of a gene you can insert? Will the efciency of your system be afected if the gene is too large? To date, the largest DNA fragment we have inserted 22 kb. Insertion efciency appears to decrease with increasing DNA fragment size. Larger fragments (>10 kb) require additional injections to obtain positive animals. in vivo Models in vivo models Bacterial Artifcial (BAC) Knock-in Mouse/Rat Models #"$VMUSBMPXDPQZWFDUPSTUIBUDBOIPMEVQUPLCPG%/"*EFBMWFDUPSTGPSJOUSPEVDUJPOPGFOUJSFHFOFTJOUPUIFSBUHFOPNF #"$USBOTHFOJDNJDFSBUTBSFTJNJMBSUPPUIFSLOPDLJONPEFMTJOUIFFYQSFTTJPOQBUUFSOPGUIFNVSJOFSBUUVTHFOFPGJOUFSFTU FYDFQU UIBUUIF#"$JTSBOEPNMZJOUFHSBUFEJOUPUIFHFOPNFBTBUSBOTHFOF"QQMJFE4UFN$FMMDBODSFBUF#"$LOPDLJOBOEDPOEJUJPOBM LOPDLJOBOJNBMNPEFMTGPSZPV

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