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site-specific recombination (SSR) technologies
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site-specific recombination technologies (SSR)

The deployment of SSR’s, i.e. Cre/CreET2, flippase (Flp/Flpe/Flpo), Dre, VCre, Vika, Nigri, Panto  & φC31,  and usage of heterotypic recombinase sites such as lox66/lox2272, Frt3/Frt5/Frt14/15, rox/rox12 creates a wealth of flexible allele design modalities for conditional mutagenesis such as cassette exchange or tight regulation of reporter gene expression through cassette inversion. Contact us to assist you in the design and construction of conditional alleles so that we may provide expertise with the following design strategies:

-conditional by inversion (COIN) as described by Economides et al, 2013¹

-flip-excision switches (FLEx) as described by Schnütgen et al, 2013² 

-invertible intronic cassette (FLIP) as described by Andersson-Rolf et al, 2017³

-Recombination mediated cassette exchange (RMCE)



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    support services

    Service

    Catalog Nr

    Service Description

    Timeline

    Deliverables

    Pricing

    • Gene editing activity testing
    • Format - cell-based transfection
    • Assay - T7 endonuclease I/Cel-II/Surveyor
     SSRM004
    NovoHelix offers a gene editing service to help clients test their CRISPR tools including guide RNAs, high-performance mutant Cas proteins and base editors in plasmid DNA or RNP formats.  A representative cell line will be transfected in triplicate and results will be generated by the mismatch-nucleases T7 Endo I or Cel-II as adopted from a protocol originally developed by Keith Joung's lab. While our cell-based assay is the gold standard for guide RNA activity, guide RNAs can be tested in an alternative in vitro cutting assay should the client prefer.  However, caution must be used in interpreting these in vitro results, as NovoHelix has broadly found that Cas9 RNP in vitro cutting of PCR amplicons as a surrogate assay for gRNA activity vastly overrepresents Cas RNP in vivo activity levels both in cellular and microinjection contexts. Hence in vitro mismatch nuclease assays often do not correlate to gene editing in vivo outcomes. In short, the cell-based gRNA testing is strongly suggested before proceeding to animal model generation.

     7-10 days

     Gene editing activity of up to 6 guide RNAs

    • Gene editing activity testing
    • Format - cell-based transfection
    • Assay - genetic reporter & flow cytometry
    SSRM005

     NovoHelix offers a gene editing service to help clients test their CRISPR tools including guide RNAs, high-performance mutant Cas proteins and base editors in plasmid DNA or RNP formats.  A representative cell line will be transfected in triplicate and results will be generated by a fluorescent reporter assay and flow cytometry.

     7-10 days

     Gene editing activity of up to 6 guide RNAs

     Genotyping Assay Development - conditional knockouts
     SSRM006
    NovoHelix offers a knockout genotyping service to help clients develop robust genotyping protocols for screening animals after breeding and expansion of indidvidual mutant/cKO founder lines.

     2-4 days

     genotyping protocol

     Development of guide RNAs - guide RNA cloning and design in a mammalian (U6, H1) expression vector or T7 in vitro transcription (T7- IVT) vector (DNA format)
    SSRM007

     Service is for design and cloning of up to 6 guide RNAs for 1 target locust.

     7-10 days

     6 guide RNAs in plasmid vectors

     Development of guide RNAs - guide RNA cloning and design with T7 in vitro transcription (T7- IVT) for use in RNP format (RNA format)
    SSRM008
     Service is for design and in vitro transcription of up to 6 guide RNAs for 1 target locust.

     7-10 days

     6 guide RNAs ~ 500 ng/ul --- RNA format

     Client-provided guide RNAs - preparation for microinjection (DNA/RNA format)
    SSRM009
    Service is for purification and for preparation of gRNAs for zygotic microinjection or slide-electroporation. Client should provide validated gRNA synthesis data such as small RNA electrophoresis results from Agilent's Bioanalyzer prior to microinjection and a minimum 200 ng/ul gRNA concentration. Purification is required to prevent the microinjection glass needle from clogging, to prevent embryo lysis and extensive delays in refitting and resetting microinjection setup.
     Client-provided dsDNA donor vector - preparation for microinjection 
    SSRM010
    Purification of supercoiled plasmid by a proprietary anion exchange (AEX) chromatography, validation by restriction digestion and analysis by gel electrophoresis. Endotoxin levels are generally very low (0.05  – 0.5 ng LPS/µg) via our proprietary extraction method and are adequate for sensitive applications such as zygotic microinjection of mammalian embryos. The plasmid is predominantly in its supercoiled topology and free of RNA and protein contamination such as RNases and proteases.  Drop dialysis in nucleofection/electroporation/microinjection buffer is included in the purification service. Validation by additional restriction digestions (greater than 3) is available with commensurate fee schedule.  Purification is compulsory to prevent the microinjection glass needle from clogging, to prevent embryo lysis and extensive delays in refitting and resetting the microinjection setup.
     cKO dsDNA donor vector construction
    SSRM011
    Isogenic cKO dsDNA donor vector construction and Sanger-sequence verified with up to 5-kb floxed insertion.  NovoHelix will provide an in silico map and provide restriction fingerprinting for plasmid structure via 3 digests. DNA is purified by a propriety AEX (anion exchange) chromotography and suitable for microinjection into mouse zygotes or nucleofection/electroporation.  The DNA is dialyzed on a membrane support in microinjection or electroporation buffer and then spun to remove particulate matter to obviate microinjection needle clogging.
     cKO dsDNA donor vector construction - complex
    SSRM012
     Isogenic cKO dsDNA donor vector construction and Sanger-sequence verified with up to 20-kb floxed insertion.  NovoHelix will provide an in silico map and provide restriction fingerprinting for plasmid structure via 3 digests. DNA is purified by a propriety AEX (anion exchange) chromotography and suitable for microinjection into mouse zygotes or nucleofection/electroporation.  The DNA is dialyzed on a membrane support in microinjection or electroporation buffer and then spun to remove particulate matter to obviate microinjection needle clogging.
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    model generation

    Service

    Catalog Nr

    Service Description

    Timeline

    Deliverables

    Pricing

    • SSR allele
    • C57BL/6 mouse via CRISPR microinjection 
    • guaranteed founders
     SSRM001

    The service includes donor vector or long single-stranded DNA (lssDNA) construction, guide RNA design using the latest design guidelines, i.e. full-length guides, chemically modified gRNAs, truncated sgRNAs, hp-sgRNAs (hairpin-sgRNAs), that produces high on-target activity in our surrogate cell-based gene editing assay while limiting off-target activity. 

     3 - 4 months founders; 5 - 8 months GLT F1s

     At least 2 SSR founders (or germline transmitted F1s) with the confirmed mutation

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    • SSR allele
    • C57BL/6 mouse via CRISPR microinjection 
    • non-guaranteed service (per injection session) 
     SSRM002

    SSR knock-in mice production with microinjection of the repair template/Cas RNP complex performed as a per session service with a total of 500 zygotes injected. The service includes donor vector or long single-stranded DNA (lssDNA) construction, guide RNA design using the latest design guidelines, i.e. full-length guides, chemically modified gRNAs, truncated sgRNAs, hp-sgRNAs (hairpin-sgRNAs), that produces high on-target activity in our surrogate cell-based gene editing assay while limiting off-target activity. 

     3 - 4 months founders; 5 - 8 months GLT F1s

     At least 500 embryos will be injected and implanted (2-3 microinjection sessions), which usually results in approximately 100 pups. Ear biopsies from all pups will be given to the investigator for genotyping. There is no guarantee that resulting pups will be gene edited and contain the SSR allele.

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    • SSR allele
    • custom mouse strain - CRISPR-mediated
    • guaranteed founders
     SSRM003

     SSR knock-in custom strain mice production via microinjection of the repair template/Cas RNP complex.  The service includes donor vector or long single-stranded DNA (lssDNA) construction, guide RNA design using the latest design guidelines, i.e. full-length guides, chemically modified gRNAs, truncated sgRNAs, hp-sgRNAs (hairpin-sgRNAs), that produces high on-target activity in our surrogate cell-based gene editing assay while limiting off-target activity. 

     3 - 4 months founders; 5 - 8 months GLT F1s

     At least 2 SSR founders (or germline transmitted F1s) with the confirmed mutation

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    technology
    references
    1: Economides AN, Frendewey D, Yang P, Dominguez MG, Dore AT, Lobov IB, Persaud T, Rojas J, McClain J, Lengyel P, Droguett G, Chernomorsky R, Stevens S, Auerbach W, Dechiara TM, Pouyemirou W, Cruz JM Jr, Feeley K, Mellis IA, Yasenchack J, Hatsell SJ, Xie L, Latres E, Huang L, Zhang Y, Pefanis E, Skokos D, Deckelbaum RA, Croll SD, Davis S, Valenzuela DM, Gale NW, Murphy AJ, Yancopoulos GD. Conditionals by inversion provide a universal method for the generation of conditional alleles. Proc Natl Acad Sci U S A. 2013 Aug 20;110(34):E3179-88. doi:10.1073/pnas.1217812110. Epub 2013 Aug 5. PubMed PMID: 23918385; PubMed Central PMCID: PMC3752204.

    2: Friede RL, Roessmann U. Chronic tonsillar herniation: an attempt at classifying chronic hernitations at the foramen magnum. Acta Neuropathol. 1976 Mar 30;34(3):219-35. PubMed PMID: 1266580.

    3: Andersson-Rolf A, Mustata RC, Merenda A, Kim J, Perera S, Grego T, Andrews K, Tremble K, Silva JC, Fink J, Skarnes WC, Koo BK. One-step generation of conditional and reversible gene knockouts. Nat Methods. 2017 Mar;14(3):287-289. doi: 10.1038/nmeth.4156. Epub 2017 Jan 30. PubMed PMID: 28135257; PubMed Central  PMCID: PMC5777571.
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