1. Engineered nucleases for targeted genome editing New perspectives for gene regulation BVL Symposium, 5-6 November 2014, Berlin Dr. Katia Pauwels Biosafety and Biotechnology Unit (SBB)

2. Method of the year - 2011 2 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)

3. 200220032004200520062007200820092010201120132012 Zebrafish Yeast Primate cells Rabbit ( ) Xenopus ( ) Drosophila C. elegans Maize Tobacco Zebrafish Rat Silkworm Mouse Sea urchin Petunia Arabidopsis Xenopus Rabbit Catfish Chlamy- domonas Butterfly Swine Yeast Zebrafish Arabidopsis Rat Drosophila Rice Tobacco Xenopus Pig Cow Cricket Silkworm Meganucleases ZFN TALEN CRISPR/Cas Soybean Pig Ciona intestinales C.Elegans Yeast Human cells Mouse cells Mouse Human cells Hamster cells Mouse cells Pig cells Human cells Rat cells Pauwels K, et al. (2014) N Biotechnol, 31(1),18-27 Site-directed nucleases (SDN) Rat

4. 4 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 masses Science 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

5. 5 Outline Function Structure and modalities for design (MNs, ZFNs, TALENs, CRISPR) Considerations for risk assessment How to improve Gene regulation

6. ZFNs TALENsMNs CRISPR NHEJ indel + donor DNA HR + donor DNA (with correction) insertion or replacement correction deletion

7. ZFNs TALENsMNs CRISPR ZFNs TALENsMNs CRISPR NHEJ Intra-chromosomal deletion ZFNs TALENsMNs CRISPRZFNs TALENsMNs CRISPR Chromosomal translocation between two different chromosomes

8. Meganucleases 8 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

9. Zinc-finger nucleases (ZFNs) 5’ 3’ 5’ 3’ 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

10. Transcription activator-like effector nucleases (TALENs) 10 5’ 3’ 5’ 3’ 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

11. CRISPR – Cas 9 11 RNA chimera PAM Target DNA 5’5’3’3’ 3’3’5’5’ 3’3’ 5’5’ 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

12. Resources for engineering 12 ZFNs TALEN CRISPR/Cas9 ZiFitTargeter Software (http://ccg.vital-it.ch/tagger/targetsearch.html )http://ccg.vital-it.ch/tagger/targetsearch.html Zinc-finger tools (http://www.scripps.edu/barbas/zfdesign/zfdesignhome.php)http://www.scripps.edu/barbas/zfdesign/zfdesignhome.php Genome-wise tag scanner for nuclease off-sites (http://ccg.vital-it.ch/tagger/targetsearch.html)http://ccg.vital-it.ch/tagger/targetsearch.html E-TALEN (http://www.e-talen.org/E-TALEN/ )http://www.e-talen.org/E-TALEN/ Genome engineering resources (http://www.genome-engineering.org/)http://www.genome-engineering.org/ Scoring algorithm for predicting TALE(N) activity (http://baolab.bme.gatech.edu/Research/BioinformaticTools/TAL_targeter.html)http://baolab.bme.gatech.edu/Research/BioinformaticTools/TAL_targeter.html ToolGen TALEN Designer (http://www.toolgen.co.kr/talen_designer/)http://www.toolgen.co.kr/talen_designer/ E-CRISP (http://www.e-crisp.org/E-CRISP/ )http://www.e-crisp.org/E-CRISP/ Genome engineering resources (http://www.genome-engineering.org/)http://www.genome-engineering.org/ RGEN tools (http://www.rgenome.net/)http://www.rgenome.net/ ZiFitTargeter Software (http://ccg.vital-it.ch/tagger/targetsearch.html )http://ccg.vital-it.ch/tagger/targetsearch.html CRISPR Design tool (http://www.broadinstitute.org/mpg/crispr_design/)http://www.broadinstitute.org/mpg/crispr_design/ Adapted from Kim and Kim, Nature reviews Genetics, 15, 321-334, 2014

13. 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

14. ModerateGoodVery good Ease of use ~1kb x 2 ~3kb x 2 4,2 kb (Cas9) + ~ 0,1 kb (sgRNA) Size of coding sequences HighPresumed high May be limited Specificity 1 in ~ 100bp 1 per bpOne per 8bp or 4bp (depending PAM) Targetable sites ZFNsTALENsCRISPR/Cas9 How to choose ?

15. 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

16. ZFNs TALENs MNs CRISPR + donor DNA NHEJ HR Gene insertion or replacement + donor DNA (with correction) Gene editing indel SDN1 SDN2 SDN3

17. 17 SDN approachEU 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

18. 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....

19. Gene regulation 19 (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

20. Gene regulation and epigenome engineering 20 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

21. 21 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