Engineered nucleases for targeted genome editing

New perspectives for gene regulation

BVL Symposium , 5-6 November 2014, Berlin

Dr. Katia PauwelsBiosafety and Biotechnology Unit (SBB)

Method of the year - 2011

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The ability to introduce targeted, tailored changes into the genomes of several species will make it feasible to ask more precise biological questions…..

Gene-editing nucleases will achieve their full potential when they can be easily and quickly designed…..

Nature Methods, Vol 9 (1) January 2012

• Meganucleases

• ZincFinger nuceases (ZFNs)

• Transcriptor Activator Like Effector Nucleases (TALENs)

2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 20132012

Zebrafish

Yeast Primate cells

Rabbit ( )

Xenopus ( )

Drosophila C. elegans

Maize Tobacco

Zebrafish

Rat

Silkworm Mouse

Sea urchinPetunia Arabidopsis

Xenopus Rabbit

Catfish Chlamy-domonas

Butterfly

Swine

Yeast

Zebrafish

Arabidopsis

Rat

Drosophila Rice

TobaccoXenopus

Pig Cow

Cricket

Silkworm

MeganucleasesZFN TALEN CRISPR/Cas

Soybean

Pig

Ciona intestinales

C.Elegans

Yeast

Human cells Human cells

Human cells Mouse cells

Mouse

Human cells

Hamster cells

Mouse cellsPig cells

Human cells

Rat cells

Pauwels K, et al. (2014) N Biotechnol, 31(1),18-27

Site-directed nucleases (SDN)

Rat

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Clustered regularly interspaced short palindromic repeat (CRISPR) – Cas system

Science, 26 September 2014 Vol 345 (6204)

“The CRISPR-Cas9 system is revolutionizing genomic engineering … this technique has grown into one of the most powerful genomic engineering tools to date”.

Genetic microsurgery of the massesScience vol 342 20 December 2013

SCIENCE - BREAKTROUGH OF THE YEAR 2013

“Such genetic microsurgery was a dream a decade ago…”

Unanticipated outcomes of basic research

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Outline

• Function

• Structure and modalities for design (MNs, ZFNs, TALENs, CRISPR)

• Considerations for risk assessment

• How to improve

• Gene regulation

ZFNsTALENs MNs

CRISPR

NHEJ

indel

+ donor DNA

HR

+ donor DNA (with correction)

insertion or replacement correction

deletion

ZFNsTALENs MNs

CRISPR ZFNsTALENs MNs

CRISPR

NHEJ

Intra-chromosomal deletion

ZFNsTALENs MNs

CRISPR ZFNsTALENs MNs

CRISPR

Chromosomal translocation between two different chromosomes

Meganucleases

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Adapted from Grizot et al., NAR, 2009, 37(16)

I-CreI endonuclease

• DNA-cleavage domain = endonuclease

• DNA-recognition domain = >12 bp DNA sequences

• no modular structure

• redesign of DNA-recognition is fastidious

Zinc-finger nucleases (ZFNs)

5’

3’ 5’

3’

FokI

FokI

• DNA-cleavage domain = FokI endonuclease

• DNA-recognition domain = Zinc Finger (ZF), each ZF binds 3bp

• ZF can be combined to recognize 9-, 12-,15-,18- bp DNA

• Important for design : heterodimeric forms , context-dependent effects

Transcription activator-like effector nucleases (TALENs)

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5’

3’ 5’

3’

FokI

FokI

LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG

Repeat variable diresidue (RVD)

• DNA-cleavage domain = FokI endonuclease

• DNA-recognition domain = tandem array of 33-35 AA repeats, each with a RVD

• One-to-one recognition : RVD module/ 1 bp

• Design : Thymine at 5’ end of target sequence, HR between RVD modules

CRISPR – Cas 9

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RNA chimera

PAMTarget DNA

5’ 3’3’ 5’

3’

5’

Cas 9crRNA tracrRNA

• DNA-cleavage system = Cas 9 nuclease

• DNA-recognition = RNA-DNA base pairing

• Target requires 2bp adjacent to region of homology (PAM)

• Multiplexed targeting by Cas9

Resources for engineering

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ZFNs

TALEN

CRISPR/Cas9

• ZiFitTargeter Software (http://ccg.vital-it.ch/tagger/targetsearch.html )• Zinc-finger tools (http://www.scripps.edu/barbas/zfdesign/zfdesignhome.php)• Genome-wise tag scanner for nuclease off-sites (http://ccg.vital-it.ch/tagger/targetsearch.html)

• E-TALEN (http://www.e-talen.org/E-TALEN/ )• Genome engineering resources (http://www.genome-engineering.org/)• Scoring algorithm for predicting TALE(N) activity (http://baolab.bme.gatech.edu/Research/BioinformaticTools/TAL_targeter.html) • ToolGen TALEN Designer (http://www.toolgen.co.kr/talen_designer/)

• E-CRISP (http://www.e-crisp.org/E-CRISP/ )• Genome engineering resources (http://www.genome-engineering.org/)• RGEN tools (http://www.rgenome.net/)• ZiFitTargeter Software (http://ccg.vital-it.ch/tagger/targetsearch.html )• CRISPR Design tool (http://www.broadinstitute.org/mpg/crispr_design/)

Adapted from Kim and Kim, Nature reviews Genetics, 15, 321-334, 2014

Improvements

Lowering Off-target activity

• In silico identification of possible genomic targets

• Understanding in vivo selectivity (target specificity) of nucleases

• Shortening the time of activity

• Avoiding homodimerization

• Lowering DNA-binding enenergy

• Nickases creating SSB on opposing strands

Versatility

Cost-effectiveness

Targetability

• Knowledge of cell’s DNA repair mechanism Reliability

Moderate Good Very good Ease of use

~1kb x 2 ~3kb x 2

4,2 kb (Cas9) + ~ 0,1 kb (sgRNA)

Size of coding sequences

High Presumed high

May be limitedSpecificity

1 in ~ 100bp

1 per bp One per 8bp or 4bp (depending PAM)

Targetable sites

ZFNs TALENs CRISPR/Cas9

How to choose ?

Methods of delivery

• Plasmid DNA (electroporation or liposome transfection)

• in vitro transcribed mRNA (micro-injection)

• Non-integrating viral vectors (IDLVs, AAVs for in vitro and in vivo delivery)

• Integrating viral vectors (LV) for continuous expression

Delivery of nucleic acids

• Recombinant proteins (e.g. ZFN, Cas9 protein complexed with gRNA)

Delivery of proteins

ZFNsTALENs MNs

CRISPR

+ donor DNA

NHEJHR

Gene insertion or replacement

+ donor DNA (with correction)

Gene editing

indel

SDN1

SDN2

SDN3

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SDN approach EU regulatory considerations Coverage by EU GMO legislation ?

 

SDN1

(indel)

 

• Excluded : mutagenesis by chemical

mutagens or ionizing radiation => so could

GMO generated by SDN1

 

No

SDN 2

(gene correction,

template DNA)

• is the template DNA a recombinant DNA ? It depends

SDN 3

(gene insertion

or replacement,

donor DNA)

• Covered : Recombinant nucleic acid

techniques involving the formation of new

combinations of genetic material

Yes

unless criteria of

self-cloning are fulfilled

SDN approaches and GMO regulatory framework

Considerations for risk assessment

• More predictable than chemical and physical mutagenesis

• Lowered hazards associated to disruption of genes and/or regulatorty elements

• Junctions are predefined (avoids creation of new and unwanted ORF )

Genetic modification at predefined locus (and genomic environment) :

For GM plants developed using SDN3 lesser event-specific

data may be necessary EFSA Journal 2012;10(10):2943.

Need for assessing off-target effects ?

• might be “tolerable” for plants

• might be “less acceptable” for animals

• needs a comprehensive assessment (risk/benefit) when applied for gene therapy

It depends because off-target effects ....

Gene regulation

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• (de)methylation of DNA

• Histone modifications

Manipulating transcriptional regulation through in situ and locus-specific

Platforms for design of synthetic transcription factors

• TALE

• CRISPR/Cas

Epigenome engineering

Gene regulation and epigenome engineering

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for in vivo optical control of endogenous gene transcription

Konermann et al., Nature (2013), 500 (7463): 472-6

Investigation of causal roles of genetic and epigenetic regulation

in normal biological processes and disease states

Light-inducible transcriptional effectors (LITEs)

Redirecting the dsDNA targeting capability of CRISPR/Cas9 for RNA-guided ssRNA binding and/or cleavage

O’Connell et al., Nature (2014), doi:10.1038

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Conclusions

• SDN and effectors have the potential to change the (epi-)genetic landscape

• Room of improvement (trade-off between activity and specificty)

• Need for in vivo off-target activity identification methods

• Safety assessment of the resulting organisms should take into account the nature of application and the intended use