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Dr Andrew Wood
University of Edinburgh
Galton Institute Advance in Genetics Conference
June 28th 2017
Genome editing:
Principles, Current and Future Uses
Genome editing defined:
Wikipedia June 2017:
…a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of a living organism using engineerednucleases
DNA cleavage DNA repair
Why is genome editing important?
• Research• Tools to study gene and genome function
• Industry:• GM plants, livestock, microbes
• Medicine• Somatic gene therapy• Animal and cellular models of disease
Talk overview
• Principles: How does genome editing work?
• Utilities: What does genome editing make possible?
• Prospects: How might genome editing be used in the future?
• Challenges: What obstacles currently prevent more widespread use?
A minimal gene editing nuclease
DNA binding module Endonuclease module
The ability of proteins to recognise specific nucleotide sequences underpins all life:
Example 1:
Transcriptional regulation:(e.g. zinc fingers, TALEs)
PromoterPromoter Protein coding sequenceProtein coding sequence
How is site-specific targeting achieved?
The ability of proteins to recognise specific nucleotide sequences underpins all life:
Example 2:
Immune recognition:
Viral DNA recognised and cleaved by host cell proteins (CRISPR)
Bacterial cellBacterial cell
Virus
Cas9
How is site-specific targeting achieved?
Zinc Fingers TAL Effectors Cas9
Era: 1990s - 2012 2010 - 2014 2013 - …
Engineered DNA binding modules
Design: Hard Easy Easy
Synthesis: Hard Hard Easy
CRISPR / Cas9
• Guide RNA = crRNA + tracrRNA
• Target specificity through watson-crick base pairing (RNA/DNA)
Double Strand Breaks (DSBs): a gateway to sequence manipulation
DSBs occur naturally (~10 – 100 / cell / day)
DSBs can be highly toxic
Elaborate mechanisms exist to sense and repair DSBs
DSB repair via end-joining
DSB repair via end-joining
DSB repair via end-joining
DSB repair via end-joining
G
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G
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A
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A
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G
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C
G
Δ 2 nucleotides
T
A
A
T
A
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T
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DSB repair via end-joining
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G
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G N S N
G N STOP Gene Knockout
Null mutation
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C
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T
A
A
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A
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nucleotide
Amino acid
nucleotide
Amino acid
DSB repair via end-joining
etc…
End joining yields deletions and insertions of variable length
Genome Editing, or Genome Vandalism?
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Homology Directed Repair allows precise sequence correction
C C A T T A G C G G A G
Target Site
Repair Template
Homology Directed Repair allows precise sequence correction
C C A T T A G C G G A G
Target Site
Repair Template
Target Site
Repair Template
C C A T T A G C G G A G
Homology Directed Repair allows precise sequence correction
C C A T T A G C G G A G
Homology Directed Repair allows precise sequence correction
Homology Directed Repair allows precise sequence correction
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G N S N
G N A N
Before
After
nucleotide
Amino acid
nucleotide
Amino acid
Precise correction is rare in primary cells
End-joining Homology-dependent repair
Deletions PRECISE GENE CORRECTION
Typically fewer than 5% of DNA breaks are repaired perfectly from external templates
Precise correction is rare in primary cells
NHEJ HDR
PRECISE GENE CORRECTION
If we understand DNA repair, we should be able to manipulate it
Deletions
Current uses of genome editing
Genome editing has ALREADY revolutionised research in molecular genetics
Reverse genetics Forward genetics
Universal toolkit for reverse genetics, that should work in any organism with available genome sequence.
Genome editing has ALREADY revolutionised research in molecular genetics
Universal toolkit for reverse genetics, that should work in any organism with available genome sequence.
Current uses of genome editing
Adapted from Kiontke et al.BMC Evolutionary Biology 2011
C. sp. 9
C. briggsaeC. sp. 5C. remaneiC. sp 16
C. sp 11C. brenneriC. sp 10
C. elegansC. sp. 19
C. sp. 17
C. sp. 18
C. sp. 14
C. sp. 7
C. japonicaC. sp. 15
C. drosophilae
C. sp. 2
C. angariaC. sp. 12
C. sp. 8
C. sp. 6
C. sp. 13
C. sp. 20
C. plicata
C. sp. 1
Pristionchus pacificus
15-30 mya
Te-Wen Lo
~150 mya
The nematode phylum as a model for evolutionary biology
Ppie-1::gfp(+)::histone/+ Ppie-1::gfp(-)::histone/+
Protocols for mutagenesis were developed in the model organism C. elegans
Adapted from Kiontke et al.BMC Evolutionary Biology 2011
C. sp. 9
C. briggsaeC. sp. 5C. remaneiC. sp 16
C. sp 11C. brenneriC. sp 10
C. elegansC. sp. 19
C. sp. 17
C. sp. 18
C. sp. 14
C. sp. 7
C. japonicaC. sp. 15
C. drosophilae
C. sp. 2
C. angariaC. sp. 12
C. sp. 8
C. sp. 6
C. sp. 13
C. sp. 20
C. plicata
C. sp. 1
Pristionchus pacificus
15-30 mya
Te-Wen Lo
~150 mya
Genome editing makes studies of gene function possible in previously unstudied species
Forward Genetics
Reverse Genetics
Approaches to study gene function
CRISPR/Cas9 is scalable for high throughput screens
2. Genome wide screens
Barcoded CRISPR libraries targeting every gene in the genome several times (>105 guide RNA targets).
CRISPR libraries introduced into cultured cells – 1 target per cell
Identify cells with phenotype of interest
Identify “hits” by sequencing CRISPR barcodes in selected cells
Using CRISPR to accelerate evolution at specific sites in the genome
Mutation SelectionChange in
allele frequency
PhenotypicSelection
GenotypingMutagenesis(allelic series)
31
Phenotype A
Phenotype B
Phenotype C
Genotype A1Genotype A2Genotype A3
…
Genotype B1Genotype B2Genotype B3
…
Genotype C1Genotype C2Genotype C3
…
Manymutant cells
Using CRISPR to accelerate evolution at specific sites in the genome
5’-ACTGACTGACTGACTGGCTGACTGACTGACTGAT
5’-TGACTGACTGACTGACCGACTGACTGACTGACTG3’-ACTGACTGACTGACTGGCTGACTGACTGACTGAC
Exogeneous repair template
Missense
2. Homology directed repair
Homology Directed Repair (HDR)
5’-ACTGANNNNNNNNNNNNNNNNNNNGACTGAC
5’-TGACTGACTGACTGACTGACTGACTGACTGACTG3’-ACTGACTGACTGACTGGCTGACTGACTGACTGAC
Heterogeneous repair template
2. Homology directed repair
Multiplex Homology Directed Repair (HDR)
5’-ACTGACTGACTGACTGACTTACTGACTGACTGAT5’-ACTGACTGACTGATTGACTTACTGACTGACTGAT5’-ACTGACTGACTGATTGGCTTACTGACTGACTGAT5’-ACTGACTGACGGACTGACTGACTGACTGACTGAT5’-ACTGACTGACTGACGGACTTACTGACTGACTGAT
5’-ACTGACTGACTGATTGACTGACTGACTGACTGAT5’-ACTGACTGACTGACTGACTGGCTGACTGACTGAT
Missense
MissenseMissenseMissenseMissense
MissenseMissense
Aktipis & Nesse 2013
Predicting mechanisms of drug resistance
Tumour Resistant sub-clone
BCR-ABL bound to Imatinib
Predicting mechanisms of drug resistance
Tumour Resistant sub-clone
Drug sensitive
Drug resistant
Frequency
Deep sequence mutated sites in
drug resistant cells
Only drug resistant mutants will grow
Drug sensitiveMultiplex
EditingUnder drug
selection
Predicting mechanisms of drug resistance
Tumour 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
Future Uses: Somatic Gene Therapy
In vivo Ex vivoCells are taken from the patient
Gene is modified in the lab
Cells are transferred back into the patient
Genes are transferred into cells while still in patient
Engineering HIV resistant T cells
CD4+
T cell
CD4 CCR5
Infection No infection
CD4 CCR5
CD4+
T cell
CD4+ T cells extracted from blood
CCR5 deletion
Cells are transferred back into the patient
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 NOT be used to generate pregnancies!
Is germline editing a useful strategy to eliminate human congenital diseases?
What traits (if any) should it be acceptable to modify in the germline?
Important Questions
Off target mutagenesis
Off target mutagenesis
Off target mutagenesis
Off target mutagenesis: other considerations
• Where OT mutations need to be avoided, careful design needed
• Select guide RNAs that do not have closely matched sequences elsewhere in the genome
• Minimise the time that genomes are exposed to Cas9
• Design of “High Fidelity” Cas9 variants
Ex vivoCells are taken from the patient
Gene is modified in the lab
If safe, cells are transferred back into the patient
Off target mutagenesis: other considerations
WGS to identify OT mutations
Strategies for reducing off target mutagenesis
• For many research applications, OT mutations are tolerable with proper controls
• Derive the same mutation with two different guide RNAs
Summary
• Genome editing tools have revolutionised our ability to control gene and genome function
• More work is required to improve safety and efficiency for clinical use
• CRISPR, TALENs and ZFNs are a prime example of how basic research can have profound and unforeseen impact
Double Strand Breaks: a gateway to sequence manipulation
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Using CRISPR to accelerate evolution at specific sites in the genome
Deep mutational scan
Specific protein or DNA
Systematic localised mutagenesis
Phenotypic screening
Which amino acids / nucleotides are functional?