Genome editing: Principles, Current and Future Uses

Transcription

1 enome editing: Principles, urrent and Future Uses Dr ndrew Wood University of Edinburgh alton Institute dvance in enetics onference June 28 th 2017

2 enome editing defined: a type of genetic engineering in which DN is inserted, deleted or replaced in the genome of a living organism using engineered nucleases Wikipedia June 2017: DN cleavage DN repair

3 Why is genome editing important? Research ools to study gene and genome function Industry: M plants, livestock, microbes Medicine Somatic gene therapy nimal and cellular models of disease

4 alk overview Principles: How does genome editing work? Utilities: What does genome editing make possible? Prospects: How might genome editing be used in the future? hallenges: What obstacles currently prevent more widespread use?

5 minimal gene editing nuclease DN binding module Endonuclease module

6 How is site-specific targeting achieved? he ability of proteins to recognise specific nucleotide sequences underpins all life: Example 1: ranscriptional regulation: (e.g. zinc fingers, LEs) Promoter Protein coding sequence

7 How is site-specific targeting achieved? he ability of proteins to recognise specific nucleotide sequences underpins all life: Example 2: Immune recognition: Viral DN recognised and cleaved by host cell proteins (RISPR) Virus Bacterial cell as9

8 Engineered DN binding modules Zinc Fingers L Effectors as9 Era: 1990s Design: Hard Easy Easy Synthesis: Hard Hard Easy

9 RISPR / as9 uide RN = crrn + tracrrn arget specificity through watson-crick base pairing (RN/DN)

10 Double Strand Breaks (DSBs): a gateway to sequence manipulation DSBs occur naturally (~ / cell / day) DSBs can be highly toxic Elaborate mechanisms exist to sense and repair DSBs

11 DSB repair via end-joining DSB repair via end-joining

12 DSB repair via end-joining DSB repair via end-joining

13 Δ 2 nucleotides DSB repair via end-joining

14 N S N N SOP ene Knockout Null mutation nucleotide mino acid nucleotide mino acid DSB repair via end-joining etc

15 End joining yields deletions and insertions of variable length enome Editing, or enome Vandalism?

16 Homology Directed Repair allows precise sequence correction arget Site Repair emplate

17 Homology Directed Repair allows precise sequence correction arget Site Repair emplate

18 Homology Directed Repair allows precise sequence correction arget Site Repair emplate

19 Homology Directed Repair allows precise sequence correction

20 Homology Directed Repair allows precise sequence correction N S N N N Before fter nucleotide mino acid nucleotide mino acid

21 Precise correction is rare in primary cells ypically fewer than 5% of DN breaks are repaired perfectly from external templates End-joining Homology-dependent repair Deletions PREISE ENE ORREION

22 Precise correction is rare in primary cells If we understand DN repair, we should be able to manipulate it NHEJ HDR Deletions PREISE ENE ORREION

23 urrent uses of genome editing enome editing has LREDY revolutionised research in molecular genetics Universal toolkit for reverse genetics, that should work in any organism with available genome sequence. Reverse genetics Forward genetics

24 urrent uses of genome editing enome editing has LREDY revolutionised research in molecular genetics Universal toolkit for reverse genetics, that should work in any organism with available genome sequence.

25 he nematode phylum as a model for evolutionary biology e-wen Lo ~150 mya dapted from Kiontke et al. BM Evolutionary Biology mya. sp. 9. briggsae. sp. 5. remanei. sp 16. sp 11. brenneri. sp 10. elegans. sp. 19. sp. 17. sp. 18. sp. 14. sp. 7. japonica. sp. 15. drosophilae. sp. 2. angaria. sp. 12. sp. 8. sp. 6. sp. 13. sp. 20. plicata. sp. 1 Pristionchus pacificus

26 Protocols for mutagenesis were developed in the model organism. elegans Ppie-1::gfp(+)::histone/+ Ppie-1::gfp(-)::histone/+

27 enome editing makes studies of gene function possible in previously unstudied species e-wen Lo ~150 mya dapted from Kiontke et al. BM Evolutionary Biology mya. sp. 9. briggsae. sp. 5. remanei. sp 16. sp 11. brenneri. sp 10. elegans. sp. 19. sp. 17. sp. 18. sp. 14. sp. 7. japonica. sp. 15. drosophilae. sp. 2. angaria. sp. 12. sp. 8. sp. 6. sp. 13. sp. 20. plicata. sp. 1 Pristionchus pacificus

28 pproaches to study gene function Reverse enetics Forward enetics

29 RISPR/as9 is scalable for high throughput screens 2. enome wide screens Barcoded RISPR libraries targeting every gene in the genome several times (>10 5 guide RN targets). RISPR libraries introduced into cultured cells 1 target per cell Identify hits by sequencing RISPR barcodes in selected cells Identify cells with phenotype of interest

30 Using RISPR to accelerate evolution at specific sites in the genome Mutation Selection hange in allele frequency

31 Using RISPR to accelerate evolution at specific sites in the genome Mutagenesis (allelic series) Phenotypic Selection Phenotype enotyping enotype 1 enotype 2 enotype 3 Many mutant cells Phenotype B enotype B1 enotype B2 enotype B3 Phenotype enotype 1 enotype 2 enotype 3 31

32 Homology Directed Repair (HDR) 2. Homology directed repair Exogeneous repair template 5 - Missense

33 Multiplex Homology Directed Repair (HDR) 2. Homology directed repair Heterogeneous repair template 5 -NNNNNNNNNNNNNNNNNNN Missense Missense Missense Missense Missense Missense Missense

34 Predicting mechanisms of drug resistance umour Resistant sub-clone ktipis & Nesse 2013 BR-BL bound to Imatinib

35 Predicting mechanisms of drug resistance umour Resistant sub-clone Drug sensitive Multiplex Editing Under drug selection Drug sensitive Drug resistant Frequency Only drug resistant mutants will grow Deep sequence mutated sites in drug resistant cells

36 Predicting mechanisms of drug resistance umour Resistant sub-clone Screen new compounds to see how easily cells evolve resistance Identify drug combinations that are resistant to resistance Prioritise lead compounds based on how easily cells can evolve resistance

37 Future Uses: Somatic ene herapy In vivo Ex vivo ells are taken from the patient enes are transferred into cells while still in patient ene is modified in the lab ells are transferred back into the patient

38 Engineering HIV resistant cells D4 + cells extracted from blood D4 R5 D4 R5 D4 + cell R5 deletion D4 + cell Infection ells are transferred back into the patient No infection

39 Editing the human germline? UK licenses granted for use in early human embryos (up to 7 Days) Specifically to study genes involved in early embryonic development Will NO be used to generate pregnancies! Important Questions Is germline editing a useful strategy to eliminate human congenital diseases? What traits (if any) should it be acceptable to modify in the germline?

40 Off target mutagenesis

41 Off target mutagenesis

42 Off target mutagenesis

43 Off target mutagenesis: other considerations Where O mutations need to be avoided, careful design needed Select guide RNs that do not have closely matched sequences elsewhere in the genome Minimise the time that genomes are exposed to as9 Design of High Fidelity as9 variants

44 Off target mutagenesis: other considerations Ex vivo ells are taken from the patient ene is modified in the lab If safe, cells are transferred back into the patient WS to identify O mutations

45 Strategies for reducing off target mutagenesis For many research applications, O mutations are tolerable with proper controls Derive the same mutation with two different guide RNs

46 Summary enome editing tools have revolutionised our ability to control gene and genome function RISPR, LENs and ZFNs are a prime example of how basic research can have profound and unforeseen impact More work is required to improve safety and efficiency for clinical use

47 Double Strand Breaks: a gateway to sequence manipulation

48 Using RISPR to accelerate evolution at specific sites in the genome Deep mutational scan Specific protein or DN Systematic localised mutagenesis Phenotypic screening Which amino acids / nucleotides are functional?