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ssODN Optimisation for CRISPR knock-ins
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D esalt
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Modification Concentration
Purification](https://image.slidesharecdn.com/thekeyconsiderationsofcrisprgenomeediting-150921102434-lva1-app6892/75/The-key-considerations-of-crispr-genome-editing-17-2048.jpg)
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ssODN Optimisation for CRISPR knock-ins
0 .5 1 2 4 8 1 6 3 2 6 4 1 2 8
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H R e ffic ie n c y w ith v a r y in g c o n c e n tr a tio n o f o lig o d o n o r
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ssODN Optimisation for CRISPR knock-ins
4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0
0 .0
0 .5
1 .0
1 .5
H R e ffic ie n c y u s in g s s O D N s o f d iffe r e n t le n g th s
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Efficiency(%)
Length
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H R e ffic ie n c y u s in g s s O D N s w ith v a r y in g n u m b e r s a n d p o s ito n s o f
p h o s p h tio la te p r o te c te d n u c le o tid e s
0 .5 1 2 4 8 1 6 3 2 6 4 1 2 8
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Targetingfrequency(GFP%)
D esalt
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Modification Concentration
Purification
Conclusion: Length 90 nucleotides, at least 3’ end having a PTO, HPLC purified
Use ~20 pmol/ 600ng of ssODN (1.5x105 cells)](https://image.slidesharecdn.com/thekeyconsiderationsofcrisprgenomeediting-150921102434-lva1-app6892/75/The-key-considerations-of-crispr-genome-editing-22-2048.jpg)
Uploaded byChris Thorne
The key considerations of crispr genome editing
The document discusses key considerations for CRISPR genome editing, emphasizing factors such as gene target selection, donor design, and validation strategies to improve editing success rates. It includes information on technical considerations like sequence source, off-target potential, and the impact of modifications on growth and expression. Additionally, the document underscores the importance of screening strategies and clone validation in the genome editing process.
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The key considerations of crispr genome editing
- 1. HORIZON DISCOVERY The KeyConsiderations for CRISPR Genome Editing Chris Thorne, PhD | Commercial Marketing Manager
- 2. 2 Disclaimer 2 • This Presentationdoes not constitute or form any part of an offer to sell, or invitation to purchase or apply for or enter into any contract or make any other commitment whatsoever in relation to, securities. Although reasonable care has been taken to ensure that the facts stated in this Presentation are accurate and that the opinions expressed are fair and reasonable, the contents of this Presentation have not been formally verified by Horizon Discovery plc (the “Company”) or any other person. Accordingly, no representation or warranty, expressed or implied, is made as to the fairness, accuracy, completeness or correctness of the information and opinions contained in this Presentation and no reliance should be placed on such information or opinions. Further, the information in this Presentation is not complete and is subject to updating, revision, further verification and amendment. Neither the Company, nor any of its subsidiaries, nor any of its respective members, directors, officers or employees nor any other person accepts any liability whatsoever for any loss howsoever arising from any use of such information or opinions or otherwise arising in connection with this Presentation. • Accordingly, information contained in the Presentation is being supplied to you solely for your information and may not be copied, reproduced or further distributed to any person or published in whole or in part, for any purpose. In particular, the distribution of this Presentation in certain jurisdictions may be restricted by law, and persons into whose possession this Presentation comes should inform themselves about, and observe, any such restrictions. Any failure to comply with these restrictions may constitute a violation of laws of any such jurisdiction. • This Presentation includes certain forward-looking statements, estimates and projections with respect to the anticipated future performance of Horizon Discovery plc, its products and the markets in which it operates. Forward-looking statements involve risks and uncertainties. Actual events could differ materially from those projected herein and such statements, estimates and projections reflect the various assumptions made by the Company which assumptions may or may not prove to be correct. These forward-looking statements speak only as at the date of this Presentation. The Company expressly disclaims any obligation or undertaking to disseminate any updates or revisions to any forward-looking statements contained in the Presentation to reflect any change in the Company’s expectations with regard thereto or any change in events, conditions or circumstances on which any such statements are based. • No part of this Presentation, or the fact of its distribution, should form the basis of or be relied upon in connection with any contract or commitment or investment decision whatsoever. This Presentation does not constitute a recommendation regarding the securities of the Company. • By participating in and/or accepting delivery of this Presentation you agree to be bound by the foregoing restrictions and the other terms of this disclaimer.
- 3. 3 CRISPR mediated genomeediting Exon 1 Exon 2 Exon 3 Exon Exon 2 Exon 31 CRISPR-induced DNA double-strand break Non-homologous end joining Exon 1 Homology-directed repair Exon 2 Exon 2Exon 2Exon 1 Frameshift mutation Exon 1 Knockout Knockin
- 4. 4 ... HOWEVER … CellLine Engineered cells! Genome Editing Vector Screen for clones As simple as….
- 5. 5 Cell Line Gene Target GuideChoice Guide Position Donor Design Screening Validation The Key Considerations For CRISPR Gene Editing There are a number of things to keep in mind to maximise your chances of success!
- 6. 6 Cell Line Gene Target GuideChoice Guide Position Donor Design Screening Validation The Key Considerations For CRISPR Gene Editing • Transfection/electroporation • Enrichment • Single-cell dilution
- 7. 7 Cell Line Gene Target GuideChoice Guide Position Donor Design Screening Validation The Key Considerations For CRISPR Gene Editing Normal human karyotype HeLa cell karyotype • Gene copy number • Effect of modification on growth
- 8. 8 The Key ConsiderationsFor CRISPR Gene Editing Cell Line Gene Target Guide Choice Guide Position Donor Design Screening Validation • Sequence source • Off-target potential vs predicted activity • Wild-type Cas9 or mutant nickase https://www.deskgen.com/ guidebook/wizard.html
- 9. 9 Cell Line Gene Target GuideChoice Guide Position Donor Design Screening Validation The Key Considerations For CRISPR Gene Editing • gRNA activity measurement NT Cas9 wt only 4uncut 1 52 3 gRNA 200 300 400 500 100 600 +ve 700 200 300 400 500 100 600 700
- 10. 10 The Key ConsiderationsFor CRISPR Gene Editing Cell Line Gene Target Guide Choice Guide Position Donor Design Screening Validation Knockouts • Be aware of spice variants/alternative start codons • Functional domain targeting
- 11. 11 The Key ConsiderationsFor CRISPR Gene Editing Cell Line Gene Target Guide Choice Guide Position Donor Design Screening Validation Doench et al
- 12. 12 The Key ConsiderationsFor CRISPR Gene Editing Cell Line Gene Target Guide Choice Guide Position Donor Design Screening Validation Knockouts • Be aware of spice variants/alternative start codons • Functional domain targeting Knockins • Cut site should be as close to site of knockin as possible
- 13. 13 Ran et alCell 2014
- 14. 14 The Key ConsiderationsFor CRISPR Gene Editing Cell Line Gene Target Guide Choice Guide Position Donor Design Screening Validation Knockouts • Be aware of spice variants/alternative start codons • Functional domain targeting Knockins • Cut site should be as close to site of knockin as possible Nickase • Minimise offset • D10A – 5’ overhang
- 15. 15 Cell Line Gene Target GuideChoice Guide Position Donor Design Screening Validation The Key Considerations For CRISPR Gene Editing • Donor sequence modifications • Effects on expression or splicing • Size and type of donor (AAV, oligo, plasmid) Cas9 Cut Site Genomic Sequence Donor Sequence containing mutation
- 16. 16 Cell Line Gene Target GuideChoice Guide Position Donor Design Screening Validation The Key Considerations For CRISPR Gene Editing GF PCMV Inactive GFP P P P RFP DrugR P -ve -ve DrugR GFCMV Active GFP P P Homologous recombinationHomologous recombination Fluorescent proteins Test Donors: Positive selection Length of homology Negative selection Negative and positive selection Inactive FIRE-line allele Active FIRE-Line allele Our Testing Platform - Fluorescent Indicator of Recombination Efficiency Our Testing Platform - FIRELine
- 17. 17 ssODN Optimisation forCRISPR knock-ins 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 0 .0 0 .5 1 .0 1 .5 H R e ffic ie n c y u s in g s s O D N s o f d iffe r e n t le n g th s O ligo length (N T) Efficiency(%) Length N o n e5 'P T O 3 'P T O5 + 3 P T O M u t F la n k M u t F la n k + 5 + 3 P T O3 x 5 'P T O3 x 3 'P T O 3 x 5 '+ 3 'P T O M u t F la n k + 3 x 5 '+ 3 'P T O 0 .0 0 .5 1 .0 Transfection%(RFP) H R e ffic ie n c y u s in g s s O D N s w ith v a r y in g n u m b e r s a n d p o s ito n s o f p h o s p h tio la te p r o te c te d n u c le o tid e s 0 .5 1 2 4 8 1 6 3 2 6 4 1 2 8 0 .0 0 .5 1 .0 1 .5 H R e ffic ie n c y w ith v a r y in g c o n c e n tr a tio n o f o lig o d o n o r [do no r] pm ol Targetingfrequency(GFP%) s s O D N 1 s s O D N 2 s s O D N 3 0 1 2 3 4 H R e ffic ie n c y u s in g o lig o s p u r fiie d b y d iffe r e n t m e th o d s Targetingfrequency(GFP%) D esalt P A G E H P LC Modification Concentration Purification
- 18. 18 ssODN Optimisation forCRISPR knock-ins 40 60 80 100 120 140 160 180 200 0.0 0.5 1.0 1.5 HR efficiency using ssODNs of different lengths Oligo length (NT) Efficiency(%)
- 19. 19 ssODN Optimisation forCRISPR knock-ins 0 .5 1 2 4 8 1 6 3 2 6 4 1 2 8 0 .0 0 .5 1 .0 1 .5 H R e ffic ie n c y w ith v a r y in g c o n c e n tr a tio n o f o lig o d o n o r [do no r] pm ol Targetingfrequency(GFP%)
- 20. 20 ssODN Optimisation forCRISPR knock-ins s s O D N 1 s s O D N 2 s s O D N 3 0 1 2 3 4 H R e ffic ie n c y u s in g o lig o s p u r fiie d b y d iffe r e n t m e th o d s Targetingfrequency(GFP%) D esalt P A G E H P LC
- 21. 21 ssODN Optimisation forCRISPR knock-ins HR efficiency using ssODNs with varying numbers and positions of phosphorothioate protected nucleotides N o n e5 'P T O 3 'P T O5 + 3 P T O M u t F la n k M u t F la n k + 5 + 3 P T O3 x 5 'P T O3 x 3 'P T O 3 x 5 '+ 3 'P T O M u t F la n k + 3 x 5 '+ 3 'P T O 0 .0 0 .5 1 .0 Targetingfrequency(GFP%) H R e ffic ie n c y u s in g s s O D N s w ith v a r y in g n u m b e r s a n d p o s ito n s o f p h o s p h t io la t e p r o t e c t e d n u c le o t id e s
- 22. 22 ssODN Optimisation forCRISPR knock-ins 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 0 .0 0 .5 1 .0 1 .5 H R e ffic ie n c y u s in g s s O D N s o f d iffe r e n t le n g th s O ligo length (N T) Efficiency(%) Length N o n e5 'P T O 3 'P T O5 + 3 P T O M u t F la n k M u t F la n k + 5 + 3 P T O3 x 5 'P T O3 x 3 'P T O 3 x 5 '+ 3 'P T O M u t F la n k + 3 x 5 '+ 3 'P T O 0 .0 0 .5 1 .0 Transfection%(RFP) H R e ffic ie n c y u s in g s s O D N s w ith v a r y in g n u m b e r s a n d p o s ito n s o f p h o s p h tio la te p r o te c te d n u c le o tid e s 0 .5 1 2 4 8 1 6 3 2 6 4 1 2 8 0 .0 0 .5 1 .0 1 .5 H R e ffic ie n c y w ith v a r y in g c o n c e n tr a tio n o f o lig o d o n o r [do no r] pm ol Targetingfrequency(GFP%) s s O D N 1 s s O D N 2 s s O D N 3 0 1 2 3 4 H R e ffic ie n c y u s in g o lig o s p u r fiie d b y d iffe r e n t m e th o d s Targetingfrequency(GFP%) D esalt P A G E H P LC Modification Concentration Purification Conclusion: Length 90 nucleotides, at least 3’ end having a PTO, HPLC purified Use ~20 pmol/ 600ng of ssODN (1.5x105 cells)
- 23. 23 Cell Line Gene Target GuideChoice Guide Position Donor Design Screening Validation The Key Considerations For CRISPR Gene Editing • Donor sequence modifications • Effects on expression or splicing • Size and type of donor (AAV, oligo, plasmid) (+/+) (+/-) (-/-) (KI/-) (KI/+) (KI/KI)
- 24. 24 Cell Line Gene Target GuideChoice Guide Position Donor Design Screening Validation The Key Considerations For CRISPR Gene Editing Number of cells to screen Screening strategy Modifications on different alleles Homozygous or heterozygous modifications versus mixed cultures % cells targeted
- 25. 25 The Key ConsiderationsFor CRISPR Gene Editing Targeting Efficiency ~8% AACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTG AsnValLeuValLysThrProGlnHisValLysIleThrAspPheGlyLeuAlaLysLeu AACGTACTAGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCGGGCCAAACTG AsnValLeuValLysThrProGlnHisValLysIleThrAspPheGlyArgAlaLysLeu Clone 5 Wild-type SpeI PCR + SpeI
- 26. 26 Cell Line Gene Target GuideChoice Guide Position Donor Design Screening Validation The Key Considerations For CRISPR Gene Editing Confirmatory genotyping strategies Off-target site analysis Modification expression Contamination Heterozygous knock-in Wild type
- 27. 27 Cell Line Gene Target GuideChoice Guide Position Donor Design Screening Validation The Key Considerations For CRISPR Gene Editing Is it suitable? Is it essential/expressed/amplified? Specificity vs Efficiency Will depend on modification Donor design to maximise efficiency How many clones to find a positive? Is my engineering as expected?
- 28. 28 Next Webinar -CRISPR modified cell lines What’s possible and how they will impact your research Exon 8 Exon 9 NanoLuc polyA Exon 1 Exon 3 Translocations and Fusions Gene tagging Chromosomal deletions Chr 1 Chr 19 Point mutations Exon 8 Exon 9 *
- 29. Your Horizon Contact: t+ 44 (0)1223 655580 f + 44 (0)1223 655581 e info@horizondiscovery.com w www.horizondiscovery.com Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom Your Horizon Contact: t + 44 (0)1223 655580 f + 44 (0)1223 655581 e info@horizondiscovery.com w www.horizondiscovery.com Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom Chris Thorne, PhD Commercial Marketing Manager c.thorne@horizondiscovery.com +44 1223 204 799
Editor's Notes
- #2 Pleasure to be here to today to tell you more about Horizon and our suite of technologies based around a core expertise in human genome editing and how we are applying this to better understand the human genome, find new validated targets and support targeted drug discovery with predictive, genetically-defined, in vitro models that accurately represent target patient groups.
- #4 Generally speaking when targeting genes of interest two DNA repair pathways are used to mediate the majority of genomic modifications we want to make. The first of these is NHEJ HR
- #6 For
- #7 For
- #8 For
- #9 For
- #10 For
- #11 Knockouts Doench et al – actually position not as important as you might predict BUT if you’re aware of splice variants and alternative start codons design your guides accordingly
- #12 Knockouts Doench et al – actually position not as important as you might predict BUT if you’re aware of splice variants and alternative start codons design your guides accordingly
- #13 Knockouts Doench et al – actually position not as important as you might predict BUT if you’re aware of splice variants and alternative start codons design your guides accordingly
- #15 Knockouts Doench et al – actually position not as important as you might predict BUT if you’re aware of splice variants and alternative start codons design your guides accordingly
- #16 For
- #17 For
- #24 For
- #25 For
- #27 For
- #28 For