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Risk Analysis of Genome Editing: Laboratory Based Studies
Biological Weapons Convention - Meeting of Experts MX2
Eva Oellingrath - [email protected] - 31 July 2019, Geneva
Genome Editing
Classic genomic modifications in bacteria Induction of mutations by radiation or chemicals
First genetic modification in 1964 by Boyer & Cohen
Gene manipulation by conjugative DNA transfer
Homologous recombination
Nucleases as molecular scissors
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“Genome editing describes the specific modification of the genetic material of a living organism by deleting, replacing, or inserting a DNA sequence, typically with the aim of improving a crop or farmed
animal, or correcting a genetic disorder.” Google
BWC - Meeting of Experts MX2 Strategies for the Risk Assessment of Genome Editing
WHO article, 2019
BWC - Meeting of Experts MX2 Strategies for the Risk Assessment of Genome Editing
Zinc finger Nucleases 1996
Homing Endonucleases 2011
TALEN 2009
CRISPR/CAS 2012
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Key principle of genome editing Generation of a DNA double strand
break (DSB)
Editing by Homology Directed Repair (HDR) or Non Homologous End Joining (NHEJ)
Targeted genome modification possible by homologous recombination via HDR
Tools for genome engineering
Genome Editing
BWC - Meeting of Experts MX2 Strategies for the Risk Assessment of Genome Editing 4
Key principle of genome editing Generation of a DNA double strand
break (DSB)
Editing by Homology Directed Repair (HDR) or Non Homologous End Joining (NHEJ)
Targeted genome modification possible by homologous recombination via HDR
Genome Editing
NHEJ-mediated repair HDR-mediated repair
Nuclease-induced DSB GCTCCCAACCGCTTCAAGCTCGA CTGGGTGAAGAGCCAGTTTGCTCCCA CGAGGGTTGGCGAAGTTCGAGCT GACCCACTTCTCGGTCAAACGAGGGT
3’ 5’
5’ 3’
No DNA template
Insertion Deletion
GCTCCCAATTTGCTCCCA CGAGGGTTAAACGAGGGT
3’ 5’
5’ 3’
GCTCCCAACCATTGATGGTTTGCTCCCA CGAGGGTTGGTAACTACCAAACGAGGGT
3’ 5’
5’ 3’
GCTCCCAACCGCTTC GTTTGCTCCCA CGAGGGTTGGCGAAG CAAACGAGGGT
3’ 5’
5’ 3’
+ DNA template
Gene insertion
GCTCCCAACCGCTTC CCAGTTTGCTCCCA CGAGGGTTGGCGAAG GGTCAAACGAGGGT
3’ 5’
5’ 3’
GCTCCCAACCATTGATGGTTTGCTCCCA CGAGGGTTGGTAACTACCAAACGAGGGT
3’ 5’
5’ 3’
GCTCCCAACCATTGATGGTTTGCTCCCA CGAGGGTTGGTAACTACCAAACGAGGGT
3’ 5’
5’ 3’
GCTCCCAACCGCTTC GTTTGCTCCCA CGAGGGTTGGCGAAG CAAACGAGGGT
3’ 5’
5’ 3’
+ DNA template
Point mutation
GCTCCCAACCATTTTGCTCCCA CGAGGGTTGGTAAAACGAGGGT
3’ 5’
5’ 3’
GCTCCCAACCATTTTGCTCCCA CGAGGGTTGGTAAAACGAGGGT
3’ 5’
5’ 3’
Possible modifications Insertion
Deletion
Point Mutation
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Genome Editing
CRISPR/CAS: Breakthrough of the Year 2015
Science breakthrough of the year 2015
Based on a naturally occurring mechanism
Highly precise cleavage of genetic material
High efficiency!?
Lower costs
Ability to address any selected genomic site!?
“Simple solution for solving complex problems in genome engineering”!?
Application possible in various areas: biomedicine, biotechnology, basic research, agriculture …
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Science news stuff, 2015
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2005 1993 2012 2007 2002 1987 2013 2014
Yoshizumi Ishino discovered the CRISPR
sequence in E. coli
Francisco Mojica characterized the now called CRISPR
locus
Jansen and Mojica coined the term
CRISPR, discovery of signature cas gene
Alexander Bolotin identified the
biotechnology relevant Cas9 and the PAM
sequence
Horvath and colleagues first experimental demonstrated the CRISPR adaptive
immunity
Doudna and Charpentier, first idea of a synthetic guide
RNA as a new genome editing tool
Feng Zhang first demonstrated targeted
genome cleavage by Cas9 in human and mouse cells
first CRISPR patent was
granted to Feng Zheng
BWC - Meeting of Experts MX2 Strategies for the Risk Assessment of Genome Editing
CRISPR/CAS: Historical Development
CRISPR/CAS – Adaptive Bacterial Immune System
Adaptive bacterial immune system against viruses and foreign DNA
Found in 90% of archaea and >50% of bacteria species
Adaptive memory by assembly of foreign fragment in bacterial genome
Elimination of foreign DNA by specific recognition and cleavage
Bacterial cell
Virus Virus
Duroux-Richard, Giovannangeli, Apparailly, 2017
RNA
RNA
RNA
RNA
RNA
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Clustered Regularly Interspaced Short Palindromic Repeats/CRSIRP-associated proteins
Mohanraju et al., 2016
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• Six main CRISPR/CAS types grouped in 2 classes (~45 subtypes) • All can be found in nature in bacteria and archaea!
CRISPR/CAS9: genome editing tool
Diversity of CRISPR/CAS Systems
singleguideRNA crRNA::tracrRNA::complex
CRISPR/CAS: New Genome Engineering Technology
Cas9, ThermoFisher, 2016
Cas9
Identification of target gene Design of specific sgRNA Species specific modifications Genome editing
GIFS UNITED
Highly programmable!
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Target dsDNA
+
+
Bacterial cell Visual screening: identification of successfully edited bacteria
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Genome Editing with CRISPR/CAS in Bacteria
BWC - Meeting of Experts MX2 Strategies for the Risk Assessment of Genome Editing
Bacteria colonies on blue/white screening plate
+ HDR Fragment
Aims of My Laboratory Studies
Basic research: • Analysis of the functional regulation of an endogenous
CRISPR/CAS system in a bacterial model organism Applied research: • Evaluation of the efficiency and specificity of CRISPR/CAS9 as
tool for genome editing in bacteria • Analysis of secondary effects in selected target organism • Assessment of the misuse potential of CRISPR/CAS tools
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Basic Research: CRISPR/CAS in B. glumae
Modell organism Burkholderia glumae
Soil ß-proteobacterium
Endogenous CRISPR/CAS system
Class I multienzyme complex system
CRISPR/CAS I-F subtype
Regulation is not fully understood
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Mohanraju et al., 2016
Assessment of genome editing efficiency by statistical analysis
Determination of genome editing specificity by addressing different target regions
Different approaches and analytical methods
Comparison of traditional methods vs. CRISPR/CAS tools
Investigation in different bacterial strains
Transcriptome analysis and RT-qPCR
Phenotypic characterisation
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Applied Research: CRISPR/CAS9 Tool
BWC - Meeting of Experts MX2 Strategies for the Risk Assessment of Genome Editing
qPCR
Genome editing of different targets
Insertions of fluorescence marker
Deletion of metabolism marker
Evaluation by
Phenotypic characterisation
Metabolism activity assays
Whole genome sequencing
classcentral
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Applied Research: CRISPR/CAS9 Tool
Possible Negative Effects of CRISPR/CAS9
On-target effects Off-target effects
Additional unspecific insertion of a targeted sequence or base pairs
Additional unspecific deletion of a targeted sequence or base pairs
Target Region Target Region
X X X X X X X X
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dsDNA dsDNA
Genetic accessibility in bacteria maybe impaired by Methylation Histone-like proteins (H-NS) Secondary structure Method optimisation still required Standardised protocols Evaluation of species specific modifications Algorithms for the identification of unwanted genome edits Macmillan, 2013
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Current Limitations of Genome Editing in Bacteria
BWC - Meeting of Experts MX2 Strategies for the Risk Assessment of Genome Editing
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CRISPR/CAS biology still not well characterised
On- and Off-target effects -> unwanted gene mutations
Ease of use: extensive practical & theoretical lab knowledge still required
Efficiency and specificity must be proven case-by-case
Analysis of CRISPR/CAS-based gene manipulation requires time consuming and expensive methods (e.g. whole genome sequencing)
BWC - Meeting of Experts MX2 Strategies for the Risk Assessment of Genome Editing
Limitations of CRISPR/CAS as Genome Editing Tool
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Benefits of CRISPR/CAS as Genome Editing Tool
BWC - Meeting of Experts MX2 Strategies for the Risk Assessment of Genome Editing
Only few components required (Cas enzyme, sgRNA, DNA template…)
High DNA cleavage precision, superior efficiency and specificity
Faster than other genome editing technologies due to lesser working steps
Programmable editing tool: delete, insert or repair genes Multiplexing allows simultaneous targeting of multiple DNA sites
CRISPR/CAS Environment Human Health
Agriculture
Human gene therapy
New genetic traits
Improve crops
Revolution or
Risk?
Biofuels
Drug development/ target ID
Bioplastics
Bioremediation
Biosensing
Pesticide reduction
Nitrogen fixation
Invasive species
Vector control: Gene drives
Kevin Esvelt, modified, 2019 Wheeler, 2006
Future Applications for Genome Editing
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Misuse potential: depends on target organism, aims, capabilities
Case-by-case risk analysis required
Gene Drive technology: misuse for hostile purpose? Research on countermeasures > legitimate but problematic?
Detection of CRISPR/CAS based gene manipulation maybe difficult: attribution problems
Establishment of procedures for the responsible handling of genome editing technologies by BWC Member States
BWC - Meeting of Experts MX2 Strategies for the Risk Assessment of Genome Editing
Risks of CRISPR/CAS as Genome Editing Tool
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Biological Weapons Convention - Meeting of Experts MX2
[email protected] 31 July 2019, Geneva
Thank you!