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gene drive systems / active genetics
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gene drive systemsactive genetics

In natural or synthetic contexts, gene drives often rely on some genetic element or circuit that can selfishly copy itself from one chromosome to another chromosome typically during early development or in germ cells. This selfish copying process known as an 'active genetic' allele spreads within a population at a higher frequency than traditional Mendelian inheritance. As such, this super-Mendelian inheritance is known as the gene drive. While gene drive systems have deployed homing nucleases such as I-SceI as described by Windbichler¹ et al 2011, more recently CRISPR-mediated gene drives have been shown as a proof-of-concept to work within the mouse female germline². Building on these milestones, the gene drive technology has the potential to transform the use of rodent biomedical models by reducing the number of matings needed to generate mutant offspring of desired alleles. Because the mutant allele can spread through a population rapidly, deployment of any gene drive requires a contemporaneous biocontainment strategy to mitigate spread to nontarget populations. One solution to the wild spreading of an allele is to deploy a local daisy-chain gene drive as described by Esvelt and Church³. NovoHelix can assist the client to develop the gene drive and appropriate biocontainment strategy: please inquire with our scientific leadership team.

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model generation

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Service Description

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Pricing

  • 'active genetic' allele / CopyCat cassette
  • C57BL/6 mouse   
  • guaranteed founders 
GEDR001
NovoHelix can help the client to develop the gene drive and approriate biocontainment strategy: please inquire with our scientific leadership team.

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

 At least 2 founders (or germline transmitted F1's) with the confirmed mutation(s)

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  • 'active genetic' allele /CopyCat cassette
  • custom mouse strain 
  • guaranteed founders 
GEDR002

 NovoHelix can help the client to develop the gene drive and approriate biocontainment strategy: please inquire with our scientific leadership team.

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

 At least 2 founders (or germline transmitted F1's) with the confirmed mutation(s)

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support services

Service

Catalog Nr

Service Description

Timeline

Deliverables

Pricing

 CopyCat cassette / dsDNA donor vector construction - complex
 GEDR003
 The active genetic allele or CopyCat cassette is constructied as dsDNA donor vector and Sanger-sequence verified.  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.

 1 - 4 weeks depending on gene drive allele complexity

 5-10 micrograms of transgene plasmid; sequence validation, in silico map and 3  digests for restriction fingerprinting overall structure. 

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 Genotyping Assay Development - gene drive 
GEDR004

 NovoHelix offers a genotyping service to help clients develop robust genotyping protocols for screening animals after breeding and expansion of indidvidual mutant founder lines.

 5-7 days

 genotyping protocol

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 CopyCat cassette / dsDNA donor vector purification
GEDR005
 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.
2-3 days

 10-100 micrograms of supercoiled plasmid

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 Development of guide RNAs - guide RNA cloning and design with T7 in vitro transcription (T7- IVT) for use in RNP format (RNA format)
GEDR006

 Service is for design and in vitro transcription (IVT) of up to 6 guide RNAs for 1 target locust.

 7-10 days

 6 guide RNAs ~ 500 ng/ul

  • Gene editing activity testing
  • Format - cell-based transfection
  • Assay - T7 endonuclease I/Cel-II/Surveyor
GEDR007
 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. Guide RNAs can also be tested in an vitro cutting assay if preferred, but we have found in vitro activity to supercede in vivo activity levels both in cellular and microinjection contexts; and hence in vitro mismatch nuclease assays often do not correlate to gene editing in vivo outcomes. 
7-10 days

 Activity of up to 6 guide RNAs

  • Gene editing activity testing
  • Format - cell-based transfection
  • Assay - genetic reporter & flow cytometry
 GEDR008

 NovoHelix offers a gene or base 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

 Activity of up to 6 guide RNAs

references
Windbichler N, Menichelli M, Papathanos PA, Thyme SB, Li H, Ulge UY, Hovde BT, Baker D, Monnat RJ Jr, Burt A, Crisanti A. A synthetic homing endonuclease-based gene drive system in the human malaria mosquito. Nature. 2011 May 12;473(7346):212-5. doi: 10.1038/nature09937. Epub 2011 Apr 20. PubMed PMID:21508956; PubMed Central PMCID: PMC3093433.


Grunwald HA, Gantz VM, Poplawski G, Xu XS, Bier E, Cooper KL. Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline. Nature. 2019 Feb;566(7742):105-109. doi: 10.1038/s41586 019-0875-2. Epub 2019 Jan 23. PubMed PMID: 30675057; PubMed Central PMCID: PMC6367021.


Noble C, Min J, Olejarz J, Buchthal J, Chavez A, Smidler AL, DeBenedictis EA, Church GM, Nowak MA, Esvelt KM. Daisy-chain gene drives for the alteration of local populations. Proc Natl Acad Sci U S A. 2019 Apr 23;116(17):8275-8282. doi: 10.1073/pnas.1716358116. Epub 2019 Apr 2. PubMed PMID: 30940750; PubMed Central PMCID: PMC6486765.
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