translating genomes | personalizing medicine

Gene-editing evolved - Combining CRISPR, rAAV and ZFNs for maximum versatility and minimal hassle

Translating Genomes | Personalizing Medicine

Dr. Chris LoweR&D Director, Cell Line Engineering

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Presenter

Dr. Chris Lowe PhDR&D Director, Cell Line Engineering

Chris obtained his PhD in the field of Medical Genetics from the University of Cambridge where he engaged in research into the genetic causes of Type 1 diabetes. He joined Horizon Discovery in 2011 and has been responsible for the gene editing group since 2013.

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Content of the Presentation

Introduction of Horizon Discovery

GENESIS™ - Horizon’s precision genome editing platform

Systematic optimisation of the GENESIS™ platform

Combining CRISPR and rAAV technologies to improve

targeting efficiency

Custom cell line development service

Summary

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Translating genetic information into personalized medicines

Genomics Personalised MedicineTranslational Genomics

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Horizon Discovery’s Mission

“To translate the human genome and accelerate the discovery of personalized medicines”

Tailoring the right drugs...to the right patients...at the right time

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Genome Editing: Creating accurate genetic models

Large growth induction phenotype

Transforming alone

Milder growth induction phenotype

Non-transforming alone

Di Nicolantonio et al., PNAS, Dec. 2008Isakoff et al., Cancer Research, Jan 2006

Genome Editing: The Right Tool For The Right Outcome

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GENESIS™: The Right Tool For The Right Outcome

rAAV

• High precision / low thru-put

• Any locus, wide cell tropism

• Well validated, KI focus

• Exclusive to HD

Zinc Fingers

• Med precision / med thru-put

• Good genome coverage

• Well validated / KO Focus

• Licensed from Sigma

CRISPR

• New but high potential

• Capable of multi-gene targeting

• Simple RNA-directed cleavage

• Combinable with AAV

• Extensive IP position

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rAAV: Modify Any Genomic Loci, in Any Way, with Perfect Precision

DNA-vectors that use a natural homologous recombination (HR) in cells to alter genomic sequences

No DNA-breaks created or required (rAAV stimulates HR directly, 1000x better than plasmid vectors)

Efficient at performing all types of alterations

Wide tropism

Hard to generate multi-allelic KO’s quickly like nucleases

Ideal for ‘deep-biology’ & disease model generation

Homologous Recombination (HR) using single-stranded DNA recombinant

Adeno Associated Viruses

Nature Genetics 18, 325- 330 (1998)

rAAV: How Does It Work?

AAV = Adeno Associated Virus (ssDNA)

rAAV: What You Can Do with rAAV Gene-Editing

Point mutations/SNPs

RNAi rescue

Insertional gene disruption

Gene deletions

Long range deletions

Translocations

Amplifications

> 40 different parental cell lines now targeted; 500 projects, covering 16 tissue types

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Nuclease Methods: ZFNs

Double strand breaks are repaired by either NHEJ, or HDR in tandem with a donor

Low off target risk

High efficiencies of knockout

Reliable gene knock-outs

Double Strand Break

Non-Homologous End Joining

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Nuclease Methods: CRISPR

Analogous to ZFNs/TALENs, but much simpler: no protein engineering required

Short ‘guide’ RNAs with homology to target loci direct a generic nuclease (Cas9)

Guide RNA + Cas9 are delivered into the cell

Cas9 cleavage is repaired by either NHEJ, or HDR in tandem with a donor

High efficiencies of knockout or knock-in

Multiplexing (multiple gene KOs in parallel) possible

hCAS9

Guide RNA

+‘Nick’ or Break KO

CRISPR components delivered into cell by transfection or electroporation

CAS 9

Guide RNAPAM

sequenceMatching genomic

sequence

Genomic DNA

Donor KI

OR

Jinek M, Science 2102. Mali P, Science 2013. Cong L, Science 2013

Nuclease Methods: Cas9 Wild-Type or Cas9 Nickase?

Cas9 Wild type Cas9 Nickase (Cas9n)

Induces double strand break Only “nicks” a single strand

Only requires single gRNA Requires two guide RNAs for reasonable activity

Concerns about off-target specificity Reduced likelihood of off-target events

High efficiency of cleavage Especially good for random indels (= KO)

Guide efficiency dictated by efficiency of the weakest gRNA

Nishimasu et al Cell

Key Considerations for a Gene-Editing Experiment

Gene Target Specifics

Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation


Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation

Normal human karyotype

HeLa cell karyotype


Gene copy number

Number and nature of modified alleles

Effect of modification on growth


Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation


Transfection/electroporation

Single-cell dilution

Optimal growth conditions


Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation


Sequence source

Off-target potential

Guide proximity

Wild-type Cas9 or mutant nickase



Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation Ran et al Cell (2013)


Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation

NTCas9 wt

only4uncut 1 52 3

gRNA

200

300

400

500

100

600

+ve

700

200

300

400

500

100

600700


Number of gRNAs

gRNA activity measurement


Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation

Cas9 Cut Site

Genomic Sequence

Donor Sequence containing mutation


Donor sequence modifications

Modification effects on expression or splicing

Donor size

Type of donor (AAV, oligo, plasmid)

Selection based strategies


Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation


Number of cells to screen

Screening strategy

Modifications on different alleles

Homozygous or heterozygous modifications versus mixed cultures

% Cells Targeted


Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation

Heterozygous knock-in

Wild type


Confirmatory genotyping strategies

Off-target site analysis

Genetic drift/stability

Modification expression

Contamination


Cell Line

gRNA Design

gRNA Activity

Donor Design

Screening

Validation

How many copies?

Is it suitable?

What’s my goal? (Precision vs Efficiency)

Does my guide cut?

Have I minimised re-cutting?

How many clones to find a positive?

Is my engineering as expected?


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Horizon’s Experience + Developments: rAAV + CRISPR Combinations

Nucleases have historically been less efficient at performing user-defined KIs vs KOs

Combining rAAV with a nuclease allows very high efficiency KIs and KOs

% G

ree

n c

ells

(FA

Cs)

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Horizon’s Experience + Developments: Advances in AAV design

Novel negative selection targeting strategies• Reduce background of NHEJ integrations

‡ Targeting frequency is the number of correctly targeted colonies per 100 drug-resistant colonies screened.§ The fold increase is the targeting frequency of the ShRNA vectors divided by the targeting frequency of the no ShRNA vector(set at 1).

Gene-targeting frequencies at the CDK2 locus

Horizon’s Experience & Developments: Cas9-FOK1 dimers

Fusion of the dimerization-dependent FokI nuclease to a catalytically inactive Cas9

DNA modification requires dimerization of the Fok1 pairs

Dimerization can only occur using two closely spaced gRNAs

Improved specificity relative to Cas9n

Mutagenic frequencies at known off target sites

96 Clones Screened

28 Positive for cutting

7 Clones Sequenced

3 Clones with indelson all three alleles

Conserved exon 3 targeted

ENSEMBL

Using CRISPR to Generate Gene KOs and KIs

Case Study: Disruption of the MAPK3 gene in the A375 cell line (copy number = 3)

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2

3

Parental

Allele 1Allele 2Allele 3

Using CRISPR to Generate Gene KOs and KIs

Case Study: Disruption of the MAPK3 gene in the A375 cell line (copy number = 3)

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Applications of Horizon’s Engineered Cell Lines

Case study: DLD-1 BRCA2 null cell line:

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DLD-1 BRCA2 null cells show selective sensitivity to the PARP inhibitor olaparib

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Applications of Horizon’s Engineered Cell Lines

Case study: SW48 PI3Ka cell lines

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Resistance to a tyrosine kinase inhibitor is conferred by PTEN deletion or activating mutations of PIK3CA

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Accessing GENESIS™: Custom Cell Line Development Service

The only ‘one-stop genome editing shop’ (ZFNs, CRISPR & rAAV)

Full custom services - modify any gene/loci to your requirements

No project too tough; including inducible alterations (KI or KOs)

Extensive know-how on editing in range parental cell-lines

Continuum of price, speed and design to meet all needs

Delivery of a validated custom cell line from as low as $30,000

Horizon’s scientists are experts at all forms of gene editing and so have the experience to help guide customers towards the approach that best suits their project

Point Mutations

Gene Knockouts

Deletions

Insertions

Translocations

Amplifications

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Horizon Discovery Products & Services

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Learn more about sgRNA screening in our upcoming free webinar

Webinar:

RNAi screening in drug discovery – introducing sgRNA technologies

Tue 9th Dec at 4 pm (GMT)

Shalem et al Science 2014

Useful Resources

From Horizon

Free gRNAs in Cas9 wild type vector – www.horizondiscovery.com/guidebook

Technical manuals for working with CRISPR - http://www.horizondiscovery.com/talk-to-us/technical-manuals

In the Literature

Exploring the importance of offset and overhand for nickase - http://www.cell.com/cell/abstract/S0092-8674(13)01015-5

sgRNA whole genome screening:• Shalem et al - http://www.sciencemag.org/content/343/6166/84.short• Wang et al - http://www.sciencemag.org/content/343/6166/80.abstract

On the web

Feng Zhang on Game Changing Therapeutic Technology (Link to Feng’s Video)

Guide design - http://crispr.mit.edu/

CRISPR Google Group - https://groups.google.com/forum/#!forum/crispr

http://www.horizondiscovery.com/guidebook

http://www.horizondiscovery.com/talk-to-us/technical-manuals

http://www.cell.com/cell/abstract/S0092-8674(13)01015-5

http://www.sciencemag.org/content/343/6166/84.short

http://www.sciencemag.org/content/343/6166/80.abstract

https://www.youtube.com/watch?v=Egf7vyxe3dQ&list=PL7FyXS8Qt1RNzNyy1mDlhM16IpVezoVeq

http://crispr.mit.edu/

https://groups.google.com/forum/#!forum/crispr

Your Horizon Contact:

Horizon Discovery Ltd, Building 7100, Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom

Tel: +44 (0) 1223 655 580 (Reception / Front desk) Fax: +44 (0) 1223 862 240 Email: [email protected] Web:

www.horizondiscovery.com

Dr. Chris Lowe

R&D Director

[email protected]

+44 (0)1223 655580