GENASSIST™ CRISPR & rAAV Genome Editing Tools
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Horizon Discovery offers advanced genome editing tools, including CRISPR and RAAV technologies, to facilitate the development of personalized medicine through precise gene manipulation. Their services include free CRISPR reagents for knockout generation, extensive intellectual property access, and support for researchers via their GenAssist and Guidebook resources. The company emphasizes their unique capability to combine multiple gene editing platforms to enhance research efficiency and outcomes.
GENASSIST™ CRISPR & rAAV Genome Editing Tools
- 1. GENASSIST™ CRISPR & rAAV Genome Editing Tools Join the Program Now! Visit www.horizondiscovery.com/guidebook FREE CRISPR Reagents for Knockout Generation
- 2. 2 Horizon Discovery Ltd. 7100 Cambridge Research Park, UK Our mission “to translate the human genome and accelerate the discovery of personalised medicines” Tailoring the right drugs...to the right patients...at the right time
- 3. The opportunity: translating genetic information into personalised medicines Information is no longer a bottleneck, emphasis is shifting to the ‘what does it all mean’ Genome editing is enabling the promise of the genomic era to be realized in the form of novel therapeutics and diagnostics It involves the capability to efficiently introduce targeted alterations into any specific gene in living cells 3
- 4. GENESIS™: Comprehensive genome editing rAAV • High precision / low thru-put • Any locus, wide cell tropism • Well validated, KI focus Zinc Fingers • Med precision / med thru-put • Good genome coverage • Well validated / KO Focus CRISPR • New but high potential • Capable of multi-gene targeting • Simple RNA-directed cleavage • Combinable with AAV 4 Horizon is the only source of rAAV expertise and is uniquely capable of exploiting multiple platforms: CRISPR, ZFNs and rAAV singularly or combined Horizon’s scientists are experts at all forms of gene editing and so have the experience to help guide customers towards the approach that best suits their project
- 5. 5 Nucleases: CRISPR/ Cas9 System RNA-guided platform to introduce either a double strand DNA break or a single strand nick at a specified location in the genome. Short ‘guide’ RNAs with homology to target loci direct a generic nuclease (Cas9) Two versions of Cas9 (wt & nickase) Guide RNA + Cas9 are delivered into the cell Cas9 cleavage is repaired by either NHEJ, or HDR in tandem with a donor KO/KI efficiencies are high; specificity concerns now starting to be addressed
- 6. On the surface… Genome editing with CRISPR appears as simple as: • Identifying a gRNA target sequence • Ordering an oligo with the target sequence and cloning it into a gRNA vector • Transfecting cells with the gRNA + Cas9 ... HOWEVER … 6
- 7. Considerations when designing a gene editing experiment Gene target specifics Suitability of your cell line gRNA design gRNA activity Donor design
- 8. Considerations when designing a gene editing experiment Gene target specifics How many copies of your gene exist in your cell line? Do you need to modify all of them? Will the genetic change affect growth? Would KO of one allele and modification of the other be viable/acceptable? Suitability of your cell line gRNA design gRNA activity Donor design Normal human karyotype HeLa cell karyotype 8
- 9. Considerations when designing a gene editing experiment Gene Target Specifics Suitability of your cell line Does it transfect/electroporate well? Can the cells be single-cell diluted and recover? What are the optimal growth conditions? gRNA design gRNA activity Donor design 9
- 10. Considerations when designing a gene editing experiment Gene Target Specifics Suitability of your cell line gRNA design What sequence source are you using? What is the best guide sequence? How close is the guide to the desired mutation? What are the potential off-target considerations? Do you want to use wild-type Cas9 or the mutant nickase version? gRNA activity Donor design 10
- 11. Considerations when designing a gene editing experiment Gene Target Specifics Suitability of your cell line gRNA design gRNA activity How many gRNAs need to be tested? How will you assess activity? What activity level do you need? Donor design
- 12. Considerations when designing a gene editing experiment Gene Target Specifics Suitability of your cell line gRNA design gRNA activity Donor design What if my donor is longer than an oligo? Will expression or splicing be affected? What type of donor do I use? (oligo, plasmid, AAV?) Should I use selection to enhance recovery of targeted clones?
- 13. CRISPR and rAAV Intellectual property Horizon is committed to gaining access to the widest range of CRISPR IP possible. We have taken this approach to ensure that our customers and partners are secure in the knowledge that they have the freedom to pursue their research and commercial goals when they choose to work with us. We bring to our customers the widest breadth of IP available from any commercial source, and we currently have either already taken a license to or are in late-stage negotiations to access multiple separate CRISPR IP patent estates: We have entered into a non-exclusive license agreement with ERS Genomics Ltd (ERS) , Harvard and with the Broad Institute to access intellectual property (IP) relating to the CRISPR/Cas9 gene editing system Horizon is the only company with access to rAAV as a precise gene editing or DNA/plasmid delivery platform, we are the only company able to offer hybrid rAAV/CRISPR systems that draw from the best aspects of both approaches for far superior gene editing efficiencies.
- 14. 14 Horizon’s unique capabilities: Combining rAAV + CRISPR Technologies can be combined for improved efficiency Tested using a reporter cell-line harboring an inactivating mutation in GFP Correction donor-vector supplied either as dsPlasmid, ssDNA oligos, or ssDNA rAAV rAAV = the most efficient donor vector (50 fold) %Greencells(FACs)
- 15. 15 GENASSIST™ CRISPR and rAAV enabled gene editing Cas9 wild type, nickase and Off the Shelf cloning plasmids Guide RNA Design, Manufacturing and Validation service Donor Design, Manufacturing (Oligo, Plasmids & rAAV) Viral encapsulation (Adenoviral vectors) Gene editing expert support Cell Lines available for further gene editing • HEK293 expressing Cas9 nickase • Over 550 Genetically defined Mutant cell lines (X-MAN™)
- 16. 16 Off the Shelf Plasmid Library Horizon has a variety of off the shelf plasmids available to address all your gene editing requirements gRNA and Donor cloning vectors GFP and Puromycin selection available All-in-one gRNA(s) + Cas9 Cas9 insertion into Rosa26 Locus
- 17. Guide RNA design, cloning and validation Target identified In silico guide design Report Cloning into all-in-one vector Package into adenovirus Report and oligo(s) Report and clone(s) 17 Validation
- 18. 18 Donor design and cloning/ viral packaging Optimized processes for short (<200 bp) donors that utilize Horizon’s deep gene editing expertise to maximize your chances of success Longer donors can be provided as plasmid, rAAV or Adeno Donor for insertion of Best-in-class reporter tags HaloTag® and Nanoluc® Sequencing service K-Ras HaloTag® cells labelled with HaloTag® ligand (red) and a DNA stain (green)
- 19. 19 gUIDEbook™ Horizon Discovery partnered with Desktop Genetics to develop gUIDEbook™. Designed by experts in the field of CRISPR genome editing, gUIDEbook™ addresses the needs of any researcher using CRISPR for generating knock- outs, knock-ins, mutations or tags. State of the art scoring functions Custom scoring functions and custom genomic data Forms pairs of gRNAs for use with nickase mutant Cas9 Design a donor sequence for knock-in experiments and silencing a target cut site by possible silent mutations
- 20. 20 Expert gene editing Support Horizon scientists have created hundreds of cell lines and truly understand challenges and needs of gene editing We offer tools, reagents and cell lines have been built with our expertise Access to expert scientists that perform gene editing daily on multiple technological platforms
- 21. 21 Cell Lines available for gene editing More than 550 mutant cell lines are now accessible for further gene editing: • Most important cancer mutations available • Gene editing in a defined mutant background Cas9 D10A nickase cell line and cell generation reagents • Use HEK293 Cas9 nickase as workhorse for all your gene editing projects • Generate new Cas9 transgenic cell lines with our plasmids to target ROSA26 locus Horizon would like to license your cell lines!
- 22. FREE CRISPR Knockout Generation Program Open to all academic researchers for knock-out of human genes Free guide design using gUIDEbook™, Horizon’s in silico guide design software Free cloning into an all-in one vector expressing Cas9 wt Must let horizon know when your guide has been used to generate a cell line When possible, Horizon wants to license cell lines generated in return for a royalty Only pay cost of shipping Possibility to validate guides before receiving them 22 Join the Program Now! Visit www.horizondiscovery.com/guidebook