Making genome edits in mammalian cells
•Download as PPTX, PDF•
3 likes•1,225 views
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
Recommended
More Related Content
What's hot
What's hot (20)
Similar to Making genome edits in mammalian cells
Similar to Making genome edits in mammalian cells (20)
Recently uploaded
Recently uploaded (20)
Making genome edits in mammalian cells
- 1. HORIZON DISCOVERY Making Genome Edits In Mammalian Cells Chris Thorne, PhD | Commercial Marketing Manager
- 2. 2 Contents 1. Quick recap 2. Introducing haploid genetics 3. Observations from over 1000 knockout experiments 4. Genome editing options beyond knockouts • Knock-ins, genomic deletions, translocations, gene tagging
- 3. 3 The Opportunity: Genome Editing Genome editing is the most robust and biologically relevant method for studying how genes and mutations function in driving disease
- 4. 4 CRISPR mediated genome editing 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 Most frequently CRISPR-Cas9 is used to make either knockouts (via NHEJ mediated gene disruption) or knockins (via HDR)
- 5. 5 Cell Line Gene Target Guide Choice 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?
- 6. 6 The Challenge? Polyploid cells… e.g. Disruption of the MAPK3 gene in the A375 cell line (copy number = 3) 1 2 3 Validation of frameshift disruptions in polyploid cells is a significant bottleneck
- 7. 7 Kotecki et al. (1999) in Exp Cell Res Carette et al. (2009) in Science KBM-7 is a human cell line that is haploid for all chromosomes but chromosome 8. Thijn Brummelkamp NKI/CeMM The Solution? Haploid cells...
- 8. 8 Genotyping analysis in haploid cells Exon 1 Exon 2 Exon 3 PCR with custom primers Sanger sequencing of PCR product Mutation masked by second copy Mutation leads to knockout Diploid Haploid Both editing and validation is more efficient in haploid cells
- 9. 9 (Near-) Haploid Human Cell Lines KBM-7 Near-haploid (diploid chr8, chr15) Isolated from CML patient Myeloid lineage Suspension cells HAP1 Near-haploid (chr15) Derived from KBM-7 Fibroblast like Adherent cells eHAP Fully haploid Derived from HAP1 Patent EP 13194940.6
- 10. 10 Haploid High efficiency Unambiguous genotyping Diploid Defined copy number Knockouts Diploid/haploid: >2fold Defined mutations Diploid/haploid: >10fold Knowledge base RNA sequencing Predict suitability as cellular model Essentiality dataset Predict success rate for knockouts Advantages of haploid cells for genome editing
- 11. 11 Customer Design ProductionQuality control Packaging Shipment On-demand knockouts for any human gene in 10 weeks Production pipeline
- 12. 12 Knockout cell line collections Gene Collections Kinases, Bromodomain genes, Deubiquitinases, Ubiquitin E2 ligases, HDACs, Caspases, Rab GTPases Pathway Collections Sialylation, mTOR signaling, TNF- signaling, Autophagy, Epigenetics, DNA damage responses
- 13. 13 1500 gene targeting experiments later…
- 14. 14 Editing efficiency in human cells 0 20 40 60 80 100 120 140 160 180 200 #ofgRNAprojects Editing Efficiency (in %)
- 15. 15 Cas9-induced mutational pattern PAM 0 200 400 600 800 1000 1200 1400 1600 1800 2000 -20 -18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20 Peak at position -3
- 16. 16 Cas9-induced mutational pattern Deletions Insertions
- 17. 17 Assessment of off-target editing in clonal cell lines Off-target sites
- 18. 18 Hap1 Gene Targeting – what we‘ve learned CRISPR/Cas9 is highly efficient Mutations cluster at PAM -3 Deletions are favored over insertions Off-target editing represents a minor issue
- 19. 19 So what can we do? 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 *
- 20. 20 Exon 1 Exon 2 Exon 3 Exon Exon 2 Exon 31 Cas9-induced double-strand break Exon 2 Exon 3 Homology-directed repair (precise) Exon 1 Exon 1 Introduction of point mutations by homology-directed repair
- 21. 21 Point mutation in EGFR L858R Targeting Efficiency ~8% AACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTG AsnValLeuValLysThrProGlnHisValLysIleThrAspPheGlyLeuAlaLysLeu AACGTACTAGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCGGGCCAAACTG AsnValLeuValLysThrProGlnHisValLysIleThrAspPheGlyArgAlaLysLeu Clone 5 Wild-type SpeI PCR + SpeI Inclusion of a restriction site knockin allows rapid screening
- 22. 22 Chromosomal deletions HAP1 cells are disomic for a fragment from chromosome 15
- 23. 23 Strategy for CRISPR/Cas-mediated excision of chr15 fragment
- 24. 24 Deletion of chr15 fragment is detectable by PCR 400 clones screened 5 positive clones identified ~1% targeting efficiency Essletzbichler et al Genome Research 2014
- 25. 25 Single cell clones that carry the deletion can be isolated SKY staining of clone E9
- 26. 26 Translocations / Chromosomal Fusions Chin J Cancer. 2013 Nov;32(11):594-603
- 27. 27 Interchromosomal translocation leads to CD74-ROS1 fusion Chr 5 Chr 6 Chr 5 Chr 6 ROS1-CD74 CD74-ROS1 Translocation CD74 ROS1 ex6 ex7 Chr 5 Chr 6 Simultaneous cleavage with Cas9 ex33 ex34 ex7 ex6 Screen for fusion by PCR ex33 ex34
- 28. 28 PCR screening identifies two clones with CD74-ROS1 fusion CD74-ROS1 ROS1-CD74 A10 E4 E4 A10 ~1% Clones Tested are positive for fusion
- 29. 29 Validation of both DNA and RNA Analysis of CD74-ROS1 break point in chr6 of genomic DNA CTTACGCATACTGCTGACAGTTAAATTTAGTTGAAG-GCCTGGGGCCTCAGTTTCTGCATCAGATTCATAGAA CTTACGCATACTGCTGACAGTTAAATTTAGTTGAAG-GCCTGGGGCCTCAGTTTCTGCATCAGATTCATAGAA CTTACGCATACTGCTGACAGTTAAATTTAGTTGAAGTGCCTGGGGCCTCAGTTTCTGCATCAGATTCATAGAA Predicted 1C2 1G13 ROS1 CD74 CCTGAAGTAGAAGGTCAAAGGGCCACCCTCACAGGCTGGATTACTTAATCCCTCTCTGAAATACCCACAAT CCTGAAGTAGAAGGTCAAAGGGCCACCCTCACAGGCTGGATTACTTAATCCCTCTCTGAAATACCCACAAT CCTGAAGTAGAAGGTCAAAGGGCCACCCTC------TGGATTACTTAATCCCTCTCTGAAATACCCACAAT Predicted 1C2 1G13 CD74 ROS1 CD74-ROS1 ROS1-CD74 CD74 ROS1 CD74 exon 6 ROS1 exon 34 Analysis of expression of CD74-ROS1 fusion transcript
- 30. 30 So where next? 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 *
- 31. 31 Gene Tagging - The conventional approach Gene tagging by homology-directed repair Exon 7 Exon 8 Exon 9 polyANanoLuc Exon 7 Exon 8 Exon 9 Homology-directed repair polyANanoLuc Exon 9 Genome Homology donor Two major shortcomings: a. Low overall efficiency b. Requires the synthesis of gene-specific donor templates
- 32. 32 Gene tagging by non-homologous end joining Developed further by Thijn Brummelkamp (NKI) and Daniel Lackner (Horizon)
- 33. 33 Gene tagging by non-homologous end joining Based on generic donor cassettes flanked by tia11 guide RNA recognition sites polyANanoLuc tia11tia11 tia11 gRNAU6 Cas9
- 34. 34 Gene tagging by non-homologous end joining Based on generic donor cassettes flanked by tia11 guide RNA recognition sites Exon 7 Exon 8 Exon 9 polyANanoLuctia11 Exon 7 Exon 8 Exon 9 Generic donor cassettes Non-homologous end joining (imprecise) polyANanoLuc tia11
- 35. 35 Genotyping on pools of cells after transfection gRNA 2655 --- 2656 --- 2657 2658 --- 2659 2660 2661 --- 2662 --- --- 2663 2665 2664 2666 2667 --- 669 --- ID1 MX2 IRF9 STAT1 TAP2 CCL2 IL9 13 out of 14 pools show integration of reporter cassette in right orientation Exon 9 polyANanoLuc
- 36. 36 Single clones bearing reporter constructs Gene ID gRNA ID # clones # PCR-positive clones Integration confirmed by sequencing Editing Efficiency ID1 2655 24 3 2 8% ID1 2656 24 5 5 21% IRF9 2659 24 1 1 4% IRF9 2660 24 1 0 N/A TAP2 2663 24 0 0 N/A TAP1 2664 24 0 0 N/A CCL2 2665 24 0 0 N/A CCL2 2666 24 1 1 4% IL6 996 24 3 3 13% BUT... only one clone contained an in-frame cassette integration!
- 37. 37 Sequencing of individual clones >2655-13 AACCCCCGGGGGCCGAGGGCTGCCGGTCTCCAGGGGCAGCGGATCCATGGTCTTCACACTC >2655-17 AACCCCCGGGGGCCGAGGGCTGCCGGTCTCCAGGGGCAGCGGATCCATGGTCTTCACACTC >2656-07 CCGGTCCGGGCTCCGCTCAGCACCCTCATCCAGGGGCAGCGGATCCATGGTCTTCACACTC >2656-10 CCGGTCCGGGCTCCGCTCAGCACCCTCATCCAGGGGCAGCGGATCCATGGTCTTCACACTC >2656-11 CCGGTCCGGGCTCCGCTCAGCACCCTCATCCAGGGGCAGCGGATCCATGGTCTTCACACTC >2656-15 CCGGTCCGGGCTCCGCTCAGCACCCTCATCCAGGGGCAGCGGATCCATGGTCTTCACACTC >669-14 CTGACCCAACCACAAATGCCAGCCTGCTTCCAGGGGCAGCGGATCCATGGTCTTCACACTC >2659-08 CAGATGGAGCAGGCCTTTGCCCGATACTTCCAGGGGCAGCGGATCCATGGTCTTCACACTC >2666-10 CAGAAGTGGGTTCAGGATTCCATGGACCTCCAGGGGCAGCGGATCCATGGTCTTCACACTC >669-24 ACCACCCCTGACCCAACCACAAATGCCAGCCTGCTGCAGCGGATCCATGGTCTTCACACTC >669-12 ACCACCCCTGACCCAACCACAAATGCCAGCCTGCTGCAGCGGATCCATGGTCTTCACACTC >2656-24 CGGTCCGGGCTCCGCTCAGCACCCTCAATCCAGGGGCAGCGGATCCATGGTCTTCACACTC Genomic Sequence Cassette Sequence Precise cleavage Ligation No indels Imprecise cleavage Ligation Indels Insertion is much more precise than originally predicted
- 38. 38 Assessing off-target integration of reporter cassette HAP1 NanoLuc cell lines contain single integration events (as assessed by droplet digital PCR) Hap1 ID1-NanoLuc HAP2 DACT1-NanoLuc HCT116 HK2-NanoLuc HAP1 wt NanoLuc copy number:
- 39. 39 A DACT1-NanoLuc reporter line DACT1 expression is up-regulated in response to stimulation with Activin A 0 500 1000 1500 2000 0 2000 4000 6000 8000 10000 Relativeluninescence DACT1-NanoLuc levels Relativeluninescence Activin A (ng/ml) 0 10 10050 0 10 10050 4 h stimulation 24 h stimulation Daniel Lackner
- 40. 40 The combination of CRISPR and a haploid background lends itself to both simple and complex genomic modifications Modification Targeting Efficiency in Hap1 Knockout >40% Point Mutation ~8% Chromosomal Deletion ~1% Chromosomal Translocation ~1% NHEJ Ligation Gene Tagging Up to 21%
- 41. 41 So lets wrap up! Yes, CRISPR-Cas9 genome editing can be… Easy to design Efficient Widely applicable Flexible …so how can Horizon help? But… × Not every cell line is easy to target × Not every guide is active × Genome editing is labour intensive and will not always be successful
- 42. 42 Already available… • Knockouts for >1,500 human genes • Verified by Sanger sequencing • Two independent clones per gene available • Can be supplied with gRNA used in generation • Supplied with wild type control line $990 per cell line (Academic pricing) How can Horizon advance your research?
- 43. 43 How can Horizon advance your research? Haploid Genome Editing On Demand • Hap1 cell line background • Rapid, cost effective knockouts and genomic deletions • Custom modifications also available (knockins, translocations, tags) Custom Cell Line Engineering Service • Your cell line • Your choice of modification • Fully custom service iPSC Gene Editing Service • Knockouts, knockins, mutation corrections • You supply the iPSCs • Custom modifications in 12-18 weeks In vivo Genome Editing • Many mice and rat knockout models already available • Microinjection ready guide RNAs • Custom in vivo genome editing service also available
- 44. 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 Follow me on LinkedIn: cmcthorne
Editor's Notes
- 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.
- 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
- For
- Key to this approach is confirming that a frameshift mutation has been introduced into all copies of the gene presence. In diploid or polyploid cells this requires subcloning of the PCR products such that they can be sequenced individually. Here is an example of a MAPK3 knockout in A375 cells which contain threee copies of the gene – and where we have different frameshift mutations in each allele. This need to deconvolute and verify creates a labour intensive bottleneck for cells with multiple alleles
- 10 guide RNAs, one clone each On-target site contains frameshift mutation Off-target sites: Amplify 10 closest off-target sites in each clone by PCR Submit to Sanger sequencing
- CRISPR/Cas is revolutionizing biological research Small RNA (20bp) allows the targeting of Cas9 endonuclease to any locus in the human genome (followed by PAM motif: NGG) Double-strand break inflicted by Cas9 is repaired by NHEJ NHEJ gives rise to frameshift mutations