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

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Δ 2 nucleotides

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DSB repair via end-joining

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G N STOP Gene Knockout

Null mutation

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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

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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

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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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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

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Genotype B1Genotype B2Genotype B3

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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?